Inhibitor and stimulator of stem cell proliferation and uses thereof

ABSTRACT

Disclosed and claimed are methods for the isolation and use of stem cell modulating factors for regulating stem cell cycle and for accelerating the post-chemotherapy peripheral blood cell recovery. Also disclosed and claimed are the inhibitors and stimulators of stem cell proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 08/627,173,filed Apr. 3, 1996 and now U.S. Pat. No. 5,861,483.

FIELD OF THE INVENTION

The present invention relates to the use of modulators of stem cellproliferation for regulating stem cell cycle in the treatment of humansor animals with autoimmune diseases, aging, cancer, myelodysplasia,preleukemia, leukemia, psoriasis, acquired immune deficiency syndrome(AIDS), myelodysplastic syndromes, aplastic anemia or other diseasesinvolving hyper- or hypo-proliferative conditions, as well as the use ofsuch compounds for analgesia. The present invention also relates to amethod of treatment for humans or animals anticipating or havingundergone exposure to chemotherapeutic agents, other agents which damagecycling stem cells, or radiation exposure and for protection againstsuch agents during ex vivo treatments. Finally, the present inventionrelates to the improvement of stem cell maintenance or expansioncultures for auto- and allo-transplantation procedures or for genetransfer, as well as for in vivo treatments to improve such procedures.

BACKGROUND OF THE INVENTION

Most end-stage cells in renewing systems are short-lived and must bereplaced continuously throughout life. For example, blood cellsoriginate from a self-renewing population of multipotent hematopoieticstem cells (HSC). Hematopojetic stem cells are a subpopulation ofhematopoietic cells. Hematopoietic cells can be obtained, for example,from bone marrow, umbilical cord blood or peripheral blood (eitherunmobilized or mobilized with an agent such as G-CSF); hematopoieticcells include the stem cell population, progenitor cells, differentiatedcells, accessory cells, stromal cells and other cells that contribute tothe environment necessary for production of mature blood cells.Hematopoietic progenitor cells are a subset of stem cells which are morerestricted in their developmental potency. Progenitor cells are able todifferentiate into only one or two lineages (e.g., BFU-E and CFU-E whichgive rise only to erythrocytes or CFU-GM which give rise to granulocytesand macrophages) while stem cells (such as CFU-MIX or CFU-GEMM) cangenerate multiple lineages and/or other stem cells. Because thehematopoietic stem cells are necessary for the development of all of themature cells of the hematopoietic and immune systems, their survival isessential in order to reestablish a fully functional host defense systemin subjects treated with chemotherapy or other agents.

Hematopoietic cell production is regulated by a series of factors thatstimulate growth and differentiation of hematopoietic cells, some ofwhich, for example erythropoietin, GM-CSF and G-CSF, are currently usedin clinical practice. One part of the control network which has not beenextensively characterized, however, is the physiological mechanism thatcontrols the cycling status of stem cells (Eaves et al. Blood78:110-117, 1991; Lord, in Stem Cells (C. S. Potten, Ed.) pp 401-22,1997 (Academic Press, NY)).

Early studies by Lord and coworkers showed the existence of solubleprotein factors in normal and regenerating bone marrow extracts whichcould either inhibit or stimulate stem cell proliferation (reviewed in:Lord and Wright, Blood Cells 6:581-593, 1980; Wright and Lorimore, CellTissue Kinet. 20:191-203, 1987; Marshall and Lord, Int Rev. Cyt.167:185-261, 1996). These activities were designated stem cell inhibitor(S CT) and stem cell stimulator (SCS), respectively.

To date, no candidate SCS molecules have been purified from bone marrowextracts prepared as described by Lord et al. (reviews referencedabove). Purification of either SCS or SCI from primary sources was notaccomplished due to the difficulties inherent in an in vivo assayrequiring large numbers of irradiated mice. In an attempt to overcomethese problems Pragnell and co-workers developed an in vitro assay forprimitive hematopoietic cells (CFU-A) and screened cell lines as asource of the inhibitory activity (see Graham et al. Nature 344:442-444,1990). As earlier studies had identified macrophages as possible sourcesfor SCI (Lord et al. Blood Cells 6:581-593, 1980), a mouse macrophagecell line, J774.2, was selected (Graham et al. Nature 344:442-444,1990). The conditioned medium from this cell line was used by Graham etal. for purification; an inhibitory peptide was isolated which proved tobe identical to the previously described cytokine macrophageinflammatory protein 1-alpha (MEP1α). Receptors for MIP-1α have beencloned; like other chemokine receptors, these MIP-1α receptors areseven-transmembrane domain (or “G-linked”) receptors which are coupledto guanine nucleotide (GTP) binding proteins of the G_(inhibitory)subclass (“G_(i)”) (reviewed in Murphy, Cytokine & Growth Factor Rev.7:47-64, 1996). The “inhibitory” designation for the G_(i) subclassrefers to its inhibitory activity on adenylate cyclase.

MIP-1α was isolated from a cell line, not from primary material. WhileGraham et al. observed that antibody to MIP-1α abrogated the activity ofa crude bone marrow extract, other workers have shown that otherinhibitory activities are important. For example, Graham et al. (J. Exp.Med. 178:925-32, 1993) have suggested that TGFβ, not MIP-1α, is aprimary inhibitor of hematopoietic stem cells. Further, Eaves et al.(PNAS 90:12015-19, 1993) have suggested that both MIP-1α and TGFβ arepresent at sub optimal levels in normal bone marrow and that inhibitionrequires a synergy between the two factors.

Recently, mice have been generated in which the MIP-1α gene has beendeleted by homologous recombination (Cook et al., Science 269:1583-5,1995). Such mice have no obvious derangement of their hematopoieticsystem, calling into question the role of MIP-1α as a physiologicalregulator of stem cell cycling under normal homeostatic conditions.Similarly, although transforming growth factor beta (TGFβ) also has stemcell inhibitory activities, the long period of time it takes for stemcells to respond to this cytokine suggests that it is not the endogenousfactor present in bone marrow extracts; further, neutralizing antibodiesto TGFβ do not abolish SCI activity in bone marrow supernatants(Hamnpson et al., Exp. Hemat. 19:245-249, 1991).

Other workers have described additional stem cell inhibitory factors.Frindel and coworkers have isolated a tetrapeptide from fetal calfmarrow and from liver extracts which has stem cell inhibitory activities(Lenfant et al., PNAS 86:779-782, 1989). Paukovits et al. (Cancer Res.50:328-332, 1990) have characterized a pentapeptide which, in itsmonomeric form, is an inhibitor and, in its dimeric form, is astimulator of stem cell cycling. Other factors have also been claimed tobe inhibitory in various in vitro systems (see Wright and Pragnell inBailliere's Clinical Haematology v. 5, pp. 723-39, 1992 (BailliereTinadall, Paris); Marshall and Lord, Int Rev. Cyt. 167:185-261, 1996).

Tsyrlova et al., SU 1561261 A1, disclosed a purification process for astem cell proliferation inhibitor.

Commonly owned applications WO 94/22915 and WO96/10634 disclose aninhibitor of stem cell proliferation, and are hereby incorporated byreference in their entirety.

To date, none of these factors have been approved for clinical use.However, the need exists for effective stem cell inhibitors. The majortoxicity associated with chemotherapy or radiation treatment is thedestruction of normal proliferating cells which can result in bonemarrow suppression or gastrointestinal toxicity. An effective stem cellinhibitor will protect these cells and allow for the optimztion of thesetherapeutic regimens. Just as there is a proven need for a variety ofstimulatory cytokines (i.e., cytokines such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-14, IL-15, G-CSF, GM-CSF,erythropoietin, thrombopoietin, stem cell factor, flk2/flt3 ligand,etc., which stimulate the cycling of hematopoietic cells) depending uponthe clinical situation, so too it is likely that a variety of inhibitoryfactors will be needed to address divergent clinical needs.

Further, there is a need to rapidly reverse the activity of such aninhibitor. The original studies of Lord et al. (reviews referencedabove) demonstrated that the inhibitory activity could be reversed byaddition of the stimulatory activity. While a variety of stem cellstimulatory cytokines has been identified (see above), none has beendemonstrated to represent the activity described by Lord and coworkersas being present in bone marrow extracts and of being able to reversethe activity of the inhibitor.

Hematopoietic progenitors and stem cells primarily reside in the bonemarrow in normal adults. Under certain conditions, for examplechemotherapy or treatment with cytokines such as G-CSF, large numbers ofprogenitors and stem cells egress into the peripheral blood, a processreferred to as “mobilization” (reviewed in Simmons et al., Stem Cells 12(suppl 1): 187-202, 1994; Scheding et al. Stem Cells 12 (suppl1):203-11, 1994; Mangan, Sem. Oncology 22:202-9, 1995; Moolten, Sem.Oncology 22:271-90, 1995). Recent published data suggest that the vastmajority of mobilized progenitors are not actively in cell cycle(Roberts and Metcalf, Blood 86:1600-,1995; Donahue et al., Blood87:1644-, 1996; Siegert and Serke, Bone Marrow Trans. 17:467-1996;Uchida et al., Blood 89:465-72, 1997).

Hemoglobin is a highly conserved tetrameric protein with molecularweight of approximately 64,000 Daltons. It consists of two alpha and twobeta chains. Each chain binds a single molecule of heme(ferroprotoporphyrin IX), an iron-containing prosthetic group.Vertebrate alpha and beta chains were probably derived from a singleancestral gene which duplicated and then diverged; the two chains retaina large degree of sequence identity both between themselves and betweenvarious vertebrates (see FIG. 16A). In humans, the alpha chain clusteron chromosome 16 contains two alpha genes (alpha₁ and alpha₂) which codefor identical polypeptides, as well as genes coding for other alpha-likechains: zeta, theta and several non-transcribed pseudogenes (see FIG.16B for cDNA and amino acid sequences of human alpha chain). The betachain cluster on chromosome 11 consists of one beta chain gene andseveral beta-like genes: delta, epsilon, G gamma and A gamma, as well asat least two unexpressed pseudogenes (see FIG. 16C for cDNA and aminoacid sequences of human beta chain).

The expression of these genes varies during development. In humanhematopoiesis, which has been extensively characterized, embryonicerythroblasts successively synthesize tetramers of two zeta chains andtwo epsilon chains (Gower I), two alpha chains and two epsilon chains(Gower II) or two zeta chains and two gamma chains (Hb Portland). Asembryogenesis proceeds, the predominant form consists of fetalhemoglobin (Hb F) which is composed of two alpha chains and two gammachains. Adult hemoglobin (two alpha and two beta chains) begins to besynthesized during the fetal period; at birth approximately 50% ofhemoglobin is of the adult form and the transition is complete by about6 months of age. The vast majority of hemoglobin (approximately 97%) inthe adult is of the two alpha and two beta chain variety (Hb A) withsmall amounts of Hb F or of delta chain (Hb A₂) being detectable.

Several methods have been used to express recombinant hemoglobin chainsin E. coli and in yeast (e.g., Sessen et al., Methods Enz. 231:347-364,1994; Looker et al., Methods Enz. 231:364-374, 1994; Ogden et al.,Methods Enz. 231:374-390, 1994; Martin de Llano et al., Methods Enz.231:364-374, 1994). It has thus far not been possible to expressisolated human alpha chain in high yields by recombinant methods (e.g.,Hoffman et al., PNAS 87:8521-25, 1990; Heman et al., Biochem.31:8619-28, 1992). Apparently, the isolated alpha chain does not assumea stable conformation and is rapidly degraded in E. coli. Co-expressionof beta chain with alpha chain results in increased expression of both(Hoffman et al. and Hernan et al., op. cit.). While the alpha chain hasbeen expressed as a fusion protein with a portion of the beta chain anda factor Xa recognition site (Nagai and Thorgersen, Methods Enz.231:347-364, 1994) it is expressed as an insoluble inclusion body underthese conditions.

Both the beta chain and the alpha chain contain binding sites forhaptoglobin. Haptoglobin is a serum protein with extremely high affinityfor hemoglobin (e.g., Putnam in The Plasma Proteins—Structure, Functionand Genetic Control (F. W. Putnam, Ed.) Vol. 2, pp 1-49 (Academic Press,NY); Hwang and Greer, JBC 255:3038-3041, 1980). Haptoglobin transport tothe liver is the major catabolic pathway for circulating hemoglobin.There is a single binding site for haptoglobin on the alpha chain (aminoacids 121-127) and two on the beta chain (amino acid regions 11-25 and131-146) (Kazim and Atassi, Biochem J. 197:507-510, 1981; McCormick andAtassi, J. Prot Chem. 9:735-742, 1990).

Biologically active peptides with opiate activity have been obtained byproteolytic degradation of hemoglobin (reviewed in Karelin et al.,Peptides 16:693-697, 1995). Hemoglobin alpha chain has an acid-labilecleavage site between amino acids 94-95 (Shaeffer, J. Biol. Chem.269:29530-29536, 1994).

Kregler et al. (Exp. Hemat. 9:11-21, 1981) have disclosed thathemoglobin has an enhancing activity on mouse bone marrow CFU-Cprogenitor colonies. Such assays demonstrate effects on CFU-GM and CFU-Mprogenitor populations as opposed to stem cells such as CFU-MIX. Theauthors observed activity in both isolated alpha and beta chains ofhemoglobin. This activity was abolished by treatment withN-ethylmaleimide, which suggested to Kregler et al. that sulfhydrylgroups were necessary. This observation, coupled with the fact that thestimulatory activity was resistant to trypsin digestion, suggested toKregler et al. that the C-terminal hydrophobic domain or “core” regionwas responsible for the activity. Moqattash et al. (Acta. Haematol.92:182-186, 1994) have disclosed that recombinant hemoglobin has astimulatory effect on CFU-E, BFU-E and CFU-GM progenitor cell numberwhich is similar to that observed with hemin. Mueller et al. (Blood86:1974, 1995) have disclosed that purified adult hemoglobin stimulateserythroid progenitors in a manner similar to that of hemin.

Petrov et al. (Bioscience Reports 15:1-14, 1995) disclosed the use of a“nonidentified myelopeptide mixture” in the treatment of congenitalanemia in the W^(v)/W^(v) mouse. The mixture increased the number ofspleen colonies, especially those of the erythroid type.

Heme and hemin have been extensively examined with regard to theirinfluences on hematopoiesis (see S. Sassa, Seminars Hemat. 25:312-20,1988 and N. Abraham et al., Int. J. Cell Cloning 9:185-210, 1991 forreviews). Heme is required for the maturation of erythroblasts; invitro, hemnin (chloroferroprotoporphyrin IX—i.e., heme with anadditional chloride ion) increases the proliferation of CFU-GEMM, BFU-Eand CFU-E. Similarly, hemin increases cellularity in long-term bonemarrow cultures.

“Opiates” are substances with analgesic properties similar to morphine,the major active substance in opium. Opiates can be small organicmolecules, such as morphine and other alkaloids or synthetic compounds,or endogenous peptides such as enkephalins, endorphins, dynorphins andtheir synthetic derivatives. Endogenous opiate peptides are produced invivo from larger precursors—pre-proenkephalin A for Met- andLeu-enkephalins, pre-proopiomelanocortin for α, β, and γ endorphins, andpre-prodynorphin for dynorphins A and B, α-neoendorphin andβ-neoendorphin. In addition, peptides with opiate activity can beobtained from non-classical sources such as proteolysis or hydrolysis ofproteins such as α or β casein, wheat gluten, lactalbumin, cytochromesor hemoglobin, or from other species such as frog skin (dermorphins) orbovine adrenal medulla. Such peptides have been termed “exorphins” incontrast to the classical endorphins; they are also referred to asatypical opiate peptides (Zioudrou et al., JBC 254:2446-9, 1979; Quirionand Weiss, Peptides 4:445, 1983; Loukas et al., Biochem. 22:4567, 1983;Brantl, Eur. J. Pharm. 106:213-14, 1984; Brantl et al., Eur. J. Pharm.111:293-4, 1985; Brand et al., Eur. J. Pharm. 125:309-10, 1986; Brantland Neubert, TIPS 7:6-7,1986; Glamsta et al., BBRC 184:1060-6, 1992;Teschemacher, Handbook Exp. Pharm. 104:499-28, 1993; Petrov et al.,Bioscience Reports 15:1-14, 1995; Karelin et al., Peptides 16:693-7,1995). Other endogenous peptides, such as the Tyr-MIF-1 family, havealso been shown to have opiate activity (Reed et al., Neurosci. andBiobehav. Rev. 18:519-25, 1994).

Opiates exert their actions by binding to three main pharmacologicalclasses of endogenous opiate receptors—mu, delta, and kappa. Receptorsrepresenting each pharmacological class have been cloned and shown to beG-liked receptors coupled to G_(i) (reviewed in: Reisine and Bell, TINS16: 506-510, 1993; Uh1 et al., TINS 17:89-93, 1994: Knapp et al., FASEBJ. 9:516-525, 1995; Satoh and Minami, Pharm. Ther. 68:343-64, 1995;Kieffer, Cell. Mol. Neurobiol. 15:615-635, 1995: Reisine, Neuropharm.34:463-472, 1995; Zaki et al., Ann. Rev. Pharm. Toxicol., 36:379-401,1996).

Specific agonists and antagonists are available for each receptortype—e.g., for mu receptors (which are selectively activated by DAMGOand DALDA and selectively antagonized by CTOP and naloxonazine), forkappa receptors (which are selectively activated by GR 89696 fumarate orU-69593 and selectively antagonized by nor—binaltorphiminehydrochloride) and for delta receptors (which are selectively activatedby DADLE and DPDPE and selectively antagonized by natrindole). Inaddition, there are broad-spectrum antagonists (such as naloxone) andagonists (such as etorphine) which act on all three receptor subtypes.

Both classical and atypical opiate peptides can be chemically altered orderivatized to change their specific opiate receptor binding properties(reviewed in Hruby and Gehrig, Med. Res. Rev. 9:343-401, 1989; Schiller,Prog. Med. Chem. 28: 301-40, 1991; Teschemacher, Handbook Exp. Pharm.104:499-28, 1993; Handbook of Experimental Pharmacology, A. Hertz (Ed.)volumes 104/I and 104/II, 1993, Springer Verlag, Berlin; Karelin et al.,Peptides 16:693-7, 1995). Examples include derivatives of dermorphin(e.g., DALDA) and enkephalins (e.g., DADLE, DAMGO or DAMME). Peptideswhich do not normally bind to opiate receptors, such as somatostatin,can also be derivatized to exhibit specific opiate receptor binding(e.g., CTOP (Hawkins et al., J. Pharm. Exp. Ther. 248:73, 1989)).Analogs can also be derived from alkaloids such as morphine with alteredreceptor binding properties (e.g., heroin, codeine, hydromorphone,oxymorphone, levorphanol, levallorphan, codeine, hydrocodone, oxycodone,nalorphine, naloxone, naltrexone, buprenorphine, butanorphanol andnalbuphine); in addition, small molecules structurally unrelated tomorphine can also act on opiate receptors (e.g., meperidine and itscongeners alphaprodine, diphenoxylate and fentanyl) (see Handbook ofExperimental Pharmacology, op. cit.; Goodman and Gilman's ThePharmacological Basis of Therapeutics, 7th Ed., A. G. Gilman, L. S.Goodman, T. W. Rall and F. Murad (Eds.) 1985 Macmillan Publishing Co.NY).

The endogenous opiate peptides (enkephalins, endorphins and dynorphins)have a conserved N-terminal tetrapeptide Tyr-Gly-Gly-Phe, followed byLeu or Met and any remaining C-terminal sequence. Removal of thehydroxyl group on the N-terminal Tyr (resulting in an N-terminal Phe)results in a dramatic loss of activity for Met-enkephalin. Thesestructural data led to the “message-address” hypothesis whereby theN-terminal “message” confers biological activity while the C-terminal“address” confers specificity and enhanced potency (Chavkin andGoldstein, PNAS 78:6543-7, 1981). Exorphins generally have a Tyr-Proreplacing the N-terminal Tyr-Gly of classical opiate peptides; theproline residue is thought to confer higher stability againstaminopeptidase degradation (Shipp et al., PNAS 86: 287-, 1989; Glamstaet al., BBRC 184:1060-6, 1992).

Recently an orphan receptor (“ORL1”) was cloned by virtue of sequencerelatedness to the mu, delta and kappa opiate receptors (Mollereau etal., FEBS 341:33-38, 1994; Fukuda et al., FEBS 343:42-46, 1994; Bunzowet al., FEBS 347:284-8, 1994; Chen et al., FEBS 347:279-83, 1994; Wanget al., FEBS 348:75-79, 1994; Keith et al., Reg. Peptides 54 143-4,1994; Wick et al., Mol. Brain Res. 27: 37-44, 1994, Halford et al., J.Neuroimmun. 59:91-101, 1995). The ligand for this receptor, variouslycalled nociceptin or orphanin FQ (referred to hereafter as “nociceptin”)has been cloned and shown to be a heptadecapeptide which is derived froma larger precursor (Meunier et al., Nature 377:532-535, 1995; Reinscheidet al., Science 270:792-794, 1995). It was demonstrated to havepronociceptive, hyperalgesic functions in vivo, as opposed to classicalopiates which have analgesic properties. Nociceptin has aPhe-Gly-Gly-Phe . . . N-terminal motif in contrast to theTyr-Gly-Gly-Phe . . . N-terminal motif of classical opiate peptidesdiscussed above. In keeping with the requirement for an N-terminal Tyrfor opiate activity in classical opiate peptides, nociceptin exhibitslittle or no affinity for the mu, kappa or delta opiate receptors.Similarly, the broad-spectrum opiate antagonist naloxone has noappreciable affinity for ORL1.

Enkephalins have been observed to have effects on murine hematopoiesisin vivo under conditions of immobilization stress (Goldberg et al.,Folia Biol. (Praha) 36:319-331, 1990). Leu-enkephalin inhibited andmet-enkephalin stimulated bone marrow hematopoiesis. These effects wereindirect, Goldberg et al. believed, due to effects on glucocorticoidlevels and T lymphocyte migration. Krizanac-Bengez et al. (Biomed. &Pharmacother. 46:367-43, 1992; Biomed. & Pharmacother. 49:27-31, 1995;Biomed. & Pharmacother. 50:85-91, 1996) looked at the effects of thesecompounds in vitro. Pre-treatment of murine bone marrow with Met- orLeu-enkephalin or naloxone affected the number of GM progenitor cellsobserved in a colony assay. This effect was highly variable and resultedin suppression, stimulation or no effect; further, there was no cleardose-response. This variability was ascribed by Krizanac-Bengez et al.to circadian rhythms and to accessory cells.

Recently, it has been demonstrated that mice in which the mu opiatereceptor has been deleted by homologous recombination have elevatednumbers of CFU-GM, BFU-E and CFU-GEMM per femur. Marrow and splenicprogenitors were more rapidly cycling in these mu receptor knockout micecompared to normal mice. It was not determined if these effects were dueto a direct or indirect effect on bone marrow stem cells resulting fromthe absence of the mu receptor in these animals (Broxmeyer et al., Blood88:338a, 1997).

I. Chemotherapy and Radiotherapy of Cancer

Productive research on stimulatory growth factors has resulted in theclinical use of a number of these factors (erythropoietin, G-CSF,GM-CSF, etc.). These factors have reduced the mortality and morbidityassociated with chemotherapeutic and radiation treatments. Furtherclinical benefits to patients who are undergoing chemotherapy orradiation could be realized by an alternative strategy of blockingentrance of stem cells into cell cycle thereby protecting them fromtoxic side effects. The reversal of this protection will allow for rapidrecovery of bone marrow function subsequent to chemo- or radiotherapy.

II. Bone Marrow and Stem Cell Transplantation, Ex Vivo Stem CellExpansion and Tumor Purging

Bone marrow transplantation (BMT) is a useful treatment for a variety ofhematological, autoirmune and malignant diseases. Current therapiesinclude hematopoietic cells obtained from umbilical cord blood, fetalliver or from peripheral blood (either unmobilized or mobilized withagents such as G-CSF or cyclophosphamide) as well as from bone marrow;the stem cells may be unpurified, partially purified (e.g., affinitypurification of the CD34⁺ population) or highly purified (e.g., throughfluorescent activated cell sorting using markers such as CD34, CD38 orrhodamine). Ex vivo manipulation of hematopoietic cells is currentlybeing used to expand primitive stem cells to a population suitable fortransplantation. Optimization of this procedure requires: (1) sufficientnumbers of stem cells able to maintain long term reconstitution ofhematopoiesis; (2) the depletion of graft versus host-inducingT-lymphocytes and (3) the absence of residual malignant cells. Thisprocedure can be optimized by including a stem cell inhibitor(s) and/ora stem cell stimulator(s).

The effectiveness of purging of hematopoietic cells with cytotoxic drugsin order to eliminate residual malignant cells is limited due to thetoxicity of these compounds for normal hematopoietic cells andespecially stem cells. There is a need for effective protection ofnormal cells during purging; protection can be afforded by taking stemcells out of cycle with an effective inhibitor.

III. Peripheral Stem Cell Harvesting

Peripheral blood stem cells (PBSC) offer a number of potentialadvantages over bone marrow for autologous transplantation. Patientswithout suitable marrow harvest sites due to tumor involvement orprevious radiotherapy can still undergo PBSC collections. The use ofblood stem cells eliminates the need for general anesthesia and asurgical procedure in patients who would not tolerate this well. Theapheresis technology necessary to collect blood cells is efficient andwidely available at most major medical centers. The major limitations ofthe method are both the low normal steady state frequency of stem cellsin peripheral blood and their high cycle status after mobilizationprocedures with drugs or growth factors (e.g., cyclophosphamide, G-CSF,stem cell factor). An effective stem cell inhibitor will be useful toreturn such cells to a quiescent state, thereby preventing their lossthrough differentiation.

IV. Treatment of Hyperproliferative Disorders

A number of diseases are characterized by a hyperproliferative state inwhich dysregulated stem cells give rise to an overproduction of endstage cells. Such disease states include, but are not restricted to,psoriasis, in which there is an overproduction of epidermal cells,premalignant conditions in the gastrointestinal tract characterized bythe appearance of intestinal polyps, and acquired immune deficiencysyndrome (AIDS) where early stem cells are not infected by HIV but cyclerapidly resulting in stem cell exhaustion. A stem cell inhibitor will beuseful in the treatment of such conditions.

V. Treatment of Hypoproliferative Disorders

A number of diseases are characterized by a hypoproliferative state inwhich dysregulated stem cells give rise to an underproduction of endstage cells. Such disease states include myelodysplatic syndromes oraplastic anemia, in which there is an underproduction of blood cells,and conditions associated with aging where there is a deficiency incellular regeneration and replacement A stem cell stimulator will beuseful in the treatment of such conditions.

VI. Gene Transfer

The ability to transfer genetic information into hematopoietic cells iscurrently being utilized in clinical settings. Hematopoietic cells are auseful target for gene therapy because of ease of access, extensiveexperience in manipulating and treating this tissue ex vivo and becauseof the ability of blood cells to permeate tissues. Furthermore, thecorrection of certain human genetic defects can be possible by theinsertion of a functional gene into the primitive stem cells of thehuman hematopoietic system.

There are several limitations for the introduction of genes into humanhematopoietic cells using either retrovirus vectors or physicaltechniques of gene transfer: (1) The low frequency of stem cells inhematopoietic tissues has necessitated the development of highefficiency gene transfer techniques; and (2) more rapidly cycling stemcells proved to be more susceptible to vector infection, but theincrease of the infection frequency by stimulation of stem cellproliferation with growth factors produces negative effects on long termgene expression, because cells containing the transgenes are forced todifferentiate irreversibly and lose their self-renewal. These problemscan be ameliorated by the use of a stem cell inhibitor to preventdifferentiation and loss of self-renewal and a stem cell stimulator toregulate the entry of stem cells into cycle and thereby facilitateretroviral-mediated gene transfer.

SUMMARY OF THE INVENTION

The present invention relates to compounds including peptides andpolypeptides which are inhibitors and/or stimulators of stem cellproliferation (INPROL and opiate compounds) and their use.

The present invention includes an inhibitor of stem cell proliferationcharacterized by the following properties:

(a) Specific activity (IC₅₀) less than or equal to 20 ng/ml in a murinecolony-forming spleen (CFU-S) assay (see Example 4),

(b) Molecular weight greater than 10,000 and less than 100,000 daltons(by ultrafiltration),

(c) Activity sensitive to degradation by trypsin,

(d) More hydrophobic than MIP-1α or TGFβ and separable from both byreverse phase chromatography (see Example 12),

(e) Biological activity retained after heating for one hour at 37° C.,55° C. or 75° C. in aqueous solution and

(f) Biological activity retained after precipitation with 1%hydrochloric acid in acetone.

The present invention is further characterized and distinguished fromother candidate stem cell inhibitors (e.g., MIP-1α, TGFβ and variousoligopeptides) by its capacity to achieve inhibition in an in vitroassay after a short preincubation period (see Example 5).

The present invention also comprises pharmaceutical compositionscontaining INPROL for treatment of a variety of disorders.

The present invention provides a method of treating a subjectanticipating exposure to an agent capable of killing or damaging stemcells by administering to that subject an effective amount of a stemcell inhibitory composition. The stem cells protected by this method canbe hematopoietic stem cells ordinarily present and dividing in the bonemarrow, cord blood, fetal liver or mobilized into the peripheral bloodcirculation. While the majority of mobilized stem cells are quiescentaccording to fluorescence activated cell sorter (FACS) analysis, themultipotential stem cells are demonstrated to be cycling and inhibitableby INPROL at stem cell inhibitory amounts. Alternatively, stem cells canbe epithelial, located for example, in the intestines or scalp or otherareas of the body or germ cells located in reproductive organs. Themethod of this invention can be desirably employed on humans, althoughanimal treatment is also encompassed by this method. As used herein, theterms “subject” or “patient” refer to an animal, such as a mammal,including a human.

The present invention also provides a method of treating a subject withhypoproliferating stem cells by administering to that subject aneffective amount of a stem cell stimulatory composition. The stem cellsstimulated by this method can be hematopoietic stem cells ordinarilypresent in the bone marrow, cord blood, fetal liver or mobilized intothe peripheral blood circulation; such stem cells may have previouslybeen placed into quiescence by use of INPROL at stem cell inhibitoryamounts. INPROL at stem cell stimulatory amounts will allow forstimulation of stem cell cycling when desired—for example, afterharvesting of stem cells for use during ex vivo expansion, or in vivosubsequent to stem cell transplantation and engraftment. Alternatively,stem cells can be epithelial, located for example, in the intestines, orscalp or other areas of the body or germ cells located in reproductiveorgans.

In another aspect, the invention provides a method for protecting andrestoring the hematopoietic, immune or other stem cell systems of apatient undergoing chemotherapy, which includes administering to thepatient an effective stem cell inhibitory amount of INPROL and/or tostimulate recovery after chemotherapy or radiation by administering aneffective stem cell stimulatory amount of INPROL.

In still a further aspect, the present invention involves a method foradjunctively treating any cancer, including those characterized by solidtumors (e.g., breast, colon, lung, testicular, ovarian, liver, kidney,pancreas, brain, sarcoma), by administering to a patient having canceran effective stem cell inhibitory amount of INPROL to protect stem cellsof the bone marrow, gastrointestinal tract or other organs from thetoxic effects of chemotherapy or radiation therapy and/or to stimulaterecovery after chemotherapy or radiation therapy by administering stemcell stimulatory amounts of INPROL.

Yet another aspect of the present invention involves the treatment ofleukemia (e.g., chronic myelogenous leukemia, acute myelogenousleukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia,myeloma, Hodgkin's disease), comprising treating hematopoietic cellshaving proliferating leukemia cells therein with an effective amount ofINPROL to inhibit proliferation of normal stem cells, and treating thebone marrow with a cytotoxic agent to destroy leukemia cells. Thismethod can be enhanced by the follow-up treatment of the bone marrowwith other agents that stimulate its proliferation; e.g., colonystimulating factors and/or INPROL at stem cell stimulatory amounts. Inone embodiment this method is performed in vivo. Alternatively, thismethod is also useful for ex vivo purging and expansion of hematopoieticcells for transplantation.

In still a further aspect, the method involves treating a subject havingany disorder caused by proliferating stem cells. Such disorders, such aspsoriasis, myelodysplasia, some autoimmune diseases, immuno-depressionin aging, myelodysplastic syndrome, aplastic anemia or stem cellexhaustion in AIDS are treated by administering to the subject aneffective amount of INPROL to inhibit or to stimulate proliferation ofthe stem cell in question.

The present invention provides a method for reversibly protecting stemcells from damage from a cytotoxic agent capable of killing or damagingstem cells. The method involves administering to a subject anticipatingexposure to such an agent an effective stem cell inhibitory amount ofINPROL.

The present invention also provides a method for reversibly stimulatingthe proliferation of stem cells during the recovery phase afterchemotherapy or radiation. The method involves administering to asubject anticipating exposure to such an agent, an effective stem cellstimulatory amount of INPROL.

The present invention also provides:

An inhibitor of stem cell proliferation isolated from porcine or otherbone marrow by the following procedure (see Example 12):

(a) Extraction of bone marrow and removal of particulate matter throughfiltration,

(b) Heat treatment at 56° C. for 40 minutes followed by cooling in icebath,

(c) Removal of precipitate by centrifugation at 10,000 g for 30 minutesat 4° C.,

(d) Acid precipitation by addition of supernatant to 10 volumes ofstirred ice-cold acetone containing 1% by volume concentratedhydrochloric acid and incubation at 4° C. for 16 hours,

(e) Isolation of precipitate by centrifugation at 20,000 g for 30minutes at 4° C. and washing with cold acetone followed by drying,

(f) Isolation by reverse phase chromatography and monitoring activity byinhibition of colony formation by bone marrow cells pretreated with5-fluorouracil and incubated in the presence of murine IL-3, as well asby absorption at 280 nm and by SDS-PAGE.

The present invention also provides:

A method for purifying an inhibitor of stem cell proliferationsubstantially free from other proteinaceous materials comprising thepreceding steps, as also described in more detail below.

The present invention also provides:

A method of treatment for humans or animals wherein an inhibitor of stemcell proliferation functions to ameliorate immunosuppression caused bystem cell hyperproliferation.

The present invention also provides:

A method of treatment for humans or animals wherein INPROL at stem cellstimulatory amounts ameliorates bone marrow suppression caused by stemcell hypoproliferation.

The present invention also provides:

A method of treatment for humans or animals wherein said inhibitor ofstem cell proliferation is administered after the stem cells are inducedto proliferate by exposure to a cytotoxic drug or irradiation procedure.Stem cells are normally quiescent but are stimulated to enter cell cycleafter chemotherapy. This renders them more sensitive to a secondadministration of chemotherapy; the current method protects them fromthis treatment

The present invention also provides:

A method of treatment for humans or animals wherein a stimulator of stemcell proliferation (e.g., INPROL at stem cell stimulatory amounts) isadministered, before or after INPROL at stem cell inhibitory amounts, topromote bone marrow regeneration. Stem cell inhibitory amounts of INPROLslow the rate at which stem cells transit the cell cycle and protectagainst chemotherapy or radiation; stem cell stimulatory amounts ofINPROL reverse this inhibition and promote bone marrow recovery.Conversely, stem cell stimulatory amounts of INPROL can be used topromote bone marrow recovery while stem cell inhibitory amounts are usedsubsequently to return stem cells to quiescence once bone marrowrecovery is achieved.

The present invention also provides:

A method of treatment for humans or animals wherein said inhibitor ofstem cell proliferation is administered as an adjuvant before ortogether with vaccination for the purpose of increasing immune response.

The present invention also provides:

A method of treating immune deficiency in a mammal comprisingadministering to said mammal an immunostimulatory amount of INPROL.

The present invention also provides:

A method of treating pain in a mammal comprising administering to saidmammal an analgesia-inducing amount of INPROL.

The present invention also provides:

A method of treatment for humans or animals receiving cytotoxic drugs orradiation treatment which comprises administering an effective amount ofthe inhibitor of stem cell proliferation to protect stem cells againstdamage.

The present invention also provides:

A method of treatment for humans or animals receiving cytotoxic drugs orradiation treatment which comprises administering an effective stem cellstimulatory amount of INPROL to enhance recovery after treatment.

The invention also includes a pharmaceutical composition comprisinghemoglobin and a pharmaceutically acceptible carrier.

The invention also includes a pharmaceutical composition comprising (a)a polypeptide selected from the group consisting of the alpha chain ofhemoglobin, the beta chain of hemoglobin, the gamma chain of hemoglobin,the delta chain of hemoglobin, the epsilon chain of hemoglobin and thezeta chain of hemoglobin, the polypeptide comprising amino acids 1-97 ofthe human alpha hemoglobin chain (“peptide 1-97”) and the polypeptidecomprising amino acids 1-94 of the human alpha hemoglobin chain(“peptide 1-94”) and (b) a pharmaceutically acceptible carrier. Suchpharmaceutical compositions be can composed of a single polypeptideselected from said group, a mixture of polypeptides selected from saidgroup or polypeptides from said group in the form of dimers ormultimers, with or without heme.

The invention also includes peptides having the sequences:

Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (Seq ID No:1)

(“Peptide 43-55”),

Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (Seq IDNo:2)

where the two Cys residues form a disulfide bond

(“Cyclic Peptide 43-55”),

Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys

where the two Cys residues are joined by a carbon bridge,

Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(Seq ID No:3)

(“Peptide 64-82”), and

a peptide comprising the first 97 N-terminal amino acids of human alphahemoglobin as in FIG. 16A.

Also included in the invention are proteins and peptide sequencesconsisting of modified versions of the human alpha chain which retainstem cell inhibitory and/or stimulatory properties. Such modificationsinclude, but are not limited to, removal or modification of theC-terminal hydrophobic domain (resulting in improved solubilitycharacteristics) and/or removal or modification of the haptoglobinbinding domain (resulting in improved pharmacokinetic properties). TheC-terminal hydrophobic domain in human alpha hemoglobin is comprised ofthe region from amino acids 98 (phenylalanine) to 141 (arginine) andcontains 23 hydrophobic amino acids Out of a total of 44. The entiredomain or one or more of these hydrophobic amino acids (6 alanines, 4valines, 8 leucines, 2 proline and 3 phenylalanines) can be removed bydeletion (“deleted” C-terminal hydrophobic domain). Alternatively, oneor more of these amino acids can be substituted with a non-polar aminoacid (e.g., glycine, serine, threonine, cysteine, tyrosine, asparagineor glutamine) (“substituted” C-terminal hydrophobic domain).

In another embodiment, chemical modifications such ascarboxymethylation, which decrease the hydrophobic character of thisregion and increases solubility, is used.

In another embodiment, hydrophobic residues are substituted with thecorresponding hydrophilic regions in the human beta hemoglobin sequence.For example, in the human beta hemoglobin sequence, the region betweenamino acids 107 (glycine) to 117 (histidine) or the region between aminoacids 130 (tyrosine) to 139 (asparagine) are each relatively hydrophilicand each or both can be substituted for the equivalent hydrophobicregions in human alpha hemoglobin.

The haptoglobin binding domain is contained within the C-terminalhydrophobic region and is comprised of amino acids 121-127. This regioncan be removed by deletion in its entirety or one or more amino acids inthis region can be deleted (“deleted” C-terminal haptoglobin bindingdomain). This region or one or more amino acids in this region can besubstituted with other amino acids such as, for example, poly-alanine orpoly-glycine or other amino acids which have the effect of abolishingthe binding of the polypeptide to haptoglobin but maintain the stem cellinhibitory activity (“substituted” C-terminal haptoglobin bindingdomain).

Other embodiments of the invention encompass corresponding modificationsto the beta hemoglobin chain (either in the C-terminal hydrophobicregion and/or in one or both haptoglobin binding domains (amino acids11-25 and 136-146)), and corresponding modifications to the delta,gamma, epsilon and/or zeta hemoglobin chains.

Also included in the invention are DNA sequences encoding the aboveidentified peptides, vectors containing said DNA sequences and hostcells containing said vectors. These peptides can be synthesized usingstandard chemical techniques (e.g., solid phase synthesis) or by usingrecombinant techniques (including fusion systems such as those employingglutathione-S-transferase (D. B. Smith and K. S. Johnson, Gene 67:31-40,1988), thioredoxin (LaVallie et al., Biotechnology 11:187-193, 1993) orubiquitin (Butt et al., PNAS 86:2540-4, 1989; Cherney et al., Biochem.30:10420-7, 1991; Baker et al., JBC 269:25381-6, 1994; U.S. Pat. Nos.5,132,213; 5,196,321 and 5,391,490 and PCT WO 91/17245). Each of thesearticles, applications and patents is hereby incorporated by reference.

Additionally the invention includes a method of inhibiting orstimulating stem cell proliferation comprising contacting hematopoieticcells with a compound capable of binding opiate receptors,advantageously the mu subclass of opiate receptors. Additionally theinvention includes a method of inhibiting or stimulating stem cellproliferation comprising contacting hematopoietic cells with a compoundcapable of binding nociceptin receptors (e.g., ORL1). Further, theinvention includes a method of inhibiting or stimulating stem cellproliferation comprising contacting hematopoietic cells with a compoundcapable of binding “opiate-like” receptors.

Peptides (called “hemorphins”) have been isolated from hemoglobin whichexhibit opiate activities (e.g., Brantl et al., Eur. J. Pharm,125:309-10, 1986; Davis et al. Peptides 10:747-51, 1989; Hoffman et al.,PNAS 87:8521-25, 1990; Hernan et al., Biochem. 31:8619-28, 1992; Karelinet al. Bioch. Biophys. Res. Comm, 202:410-5, 1994; Zhao et al., Ann.N.Y. Acad. Science 750:452-8, 1995; Petrov et at., Bioscience Reports,15:1-14, 1995; Karelin et al., Peptides 16:693-697, 1995). Each of thesearticles is hereby incorporated by reference. Other atypical opiatepeptides and small molecules also exist (Zioudrou et al., JBC254:2446-9,1979; Quirion and Weiss, Peptides 4:445, 1983; Loukas et al.,Biochem. 22:4567, 1983; Brantl, Eur. J. Pharm. 106:213-14, 1984; Brantlet al., Eur. J. Pharm. 111:293-4, 1985; Brand and Neubert, TIPS7:6-7,1986; Hruby and Gehrig, Med. Res. Rev. 9:343-401, 1989; Schiller,Prog. Med. Chem. 28: 301-40, 1991; Glamsta et al., BBRC 184:1060-6,1992; Teschemacher, Handbook Exp. Pharm. 104:499-28, 1993; Handbook ofExperimental Pharmacology, A. Hertz (Ed.) volumes 104/I and 104/II,1993, Springer Verlag, Berlin; Reed et al., Neurosci. and Biobehav. Rev.18:519-25, 1994; Karelin et al., Peptides 16:693-7, 1995). Each of thesearticles is hereby incorporated by reference. As used herein,“opiate-like receptors” are defined by their ability to bind opiates,INPROL, hemorphins, exorphins, nociceptin, Tyr-MIF-1 family members,alkaloids and/or other compounds which either inhibit or stimulate stemcell proliferation in a manner antagonized by the inclusion of anappropriate amount of naloxone (see Examples 29 and 38).

In addition, the invention includes a method of identifying receptor(s)and ligands comprising using INPROL (advantageously peptide forms suchas Peptide 1-94, 1-97, 43-55 or 64-82) in a receptor binding assay.Further, the invention includes a method of identifying receptor(s) andligands comprising using INPROL in an adenylate cyclase assay.

Additionally the invention includes a method of inhibiting orstimulating stem cell proliferation comprising contacting hematopoieticcells with a compound (for example, mastoparan) capable of activatingGTP-binding proteins, advantageously those of the G_(inhibitory)subtype.

The invention also includes a method of inhibiting or stimulating stemcell proliferation comprising contacting hematopoietic cells with apeptide selected from the group of hemorphin peptides having thesequence:

Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:4);

Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg, (SEQ ID NO:5);

Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:6);

Leu-Val-Val-Tyr-Pro-Trp-Thr, (SEQ ID NO:7);

Leu-Val-Val-Tyr-Pro-Trp, (SEQ ID NO:8);

Leu-Val-Val-Tyr-Pro, (SEQ ID NO:9);

Val-Val-Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:10);

Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:11);

Tyr-Pro-Trp-Thr-Gln-Arg, (SEQ ID NO:12);

Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:13); and

Tyr-Pro-Trp-Thr (SEQ ID NO:27).

The above peptides have sequence similarity and/or biological activitysimilar to other atypical opiate peptides such as those of the Tyr-MIF-1family (see Reed et al., Neurosci. Biobehav. Rev. 18:519-25, 1994 forreview), the casein-derived casomorphins (Branl et al., Hoppe-Seyler'sZ. Physiol. Chem. 360:1211-16, 1979; Loukas et al., Biochem.22:4567-4573, 1983; Fiat and Jolles, Mol. Cell. Biochem. 87:5-30, 1989),peptides derived from cytochrome b, termed cytochrophins (Brantl et al.,Eur. J. Pharm. 111:293-4, 1985), various exorphins and opiate peptidesfrom human and non-human species (Zioudrou et al., JBC 254:2446-9, 1979;Brantl, Eur. J. Pharm. 106:213-14, 1984; Branl et al., Eur. J. Pharm.125:309-10, 1986; Brantl and Neubert, TIPS 7:6-7,1986; Glamsta et al.,BBRC 184:1060-6, 1992; Teschemacher, Handbook Exp. Pharm. 104:499-28,1993; Karelin et al., Peptides 16:693-7, 1995) as well as to peptidesderived from combinatorial libraries screened for binding to opiatereceptors (see Dooley et al., Peptide Research 8:124-137, 1995 forreview). Each of these articles is hereby incorporated by reference.

The invention also includes a method of inhibiting or stimulating stemcell proliferation comprising contacting hematopoietic cells with apeptide selected from the group consisting of Tyr-MIF-1 relatedpeptides, casomorphins, cytochrophins and exorphins. Specificallyincluded are the Tyr-MIF-1 peptides having the sequences:

Tyr-Pro-Try-Gly-NH₂ (SEQ ID NO:29),

Tyr-Pro-Lys-Gly-NH₂ (SEQ ID NO:30),

Tyr-Pro-Leu-Gly-NH₂ (SEQ ID NO:31), and

Pro-Leu-Gly-NH₂.

The invention also includes a method of inhibiting or stimulating stemcell proliferation comprising contacting hematopoietic cells with anopiate peptide selected from the group consisting of

(D-Ala²,N-Me-Phe⁴,Gly-ol⁵)-Enkephalin (DAMGO),

(D-Arg², Lys⁴)-Dermorphin-(1-4)-amide (DALDA),

(Phe⁴)-Dermorphine (1-4) amide

Ac-Arg-Phe-Met-Trp-Met-Arg-NH₂, (SEQ ID NO:14);

Ac-Arg-Phe-Met-Trp-Met-Lys-NH₂, (SEQ ID NO:32); and

H-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH₂, (SEQ ID NO:33).

The invention also includes a method of inhibiting or stimulating stemcell proliferation comprising contacting hematopoietic cells with anopiate agonist compound selected from the group consisting of morphine,codeine, methadone, heroin, meperidine, alphaprodine, diphenoxylate,fentanyl, sufentanil, alfentanil, levorphanol, hydrocodone,dihydrocodeine, oxycodone, hydromorphone, propoxyphene, buprenorphine,etorphine, oxymorphone dextopropoxyphene, and meptazinol. Specificallyincluded is morphine at inhibitory amounts less than 10⁻⁷ molar.

The invention also includes a method of inhibiting or stimulating stemcell proliferation comprising contacting hematopoietic cells with anopiate antagonist or mixed agonist/antagonist selected from the groupconsisting of naloxone, naltrexone, nalorphine, pentazocine, nalbuphineand butorphanol. Specifically included is naloxone at inhibitory amountsof less than 10⁻⁸ molar.

The invention also includes a method of stimulating stem cellproliferation comprising contacting hematopoietic cells with a stem cellstimulating amount of protein or peptide selected from the group thatincludes INPROL, myoglobin, DAMGO and DALDA.

The invention also includes a method of conducting gene therapy in amammal comprising:

a) removing hematopoietic cells from said mammal,

b) treating said hematopoietic cells ex vivo with a stem cellstimulatory amount of INPROL and/or an opiate compound,

c) transfecting or infecting said hematopoietic cells with apredetermined gene,

d) contacting said transfected hematopoietic cells ex vivo with a stemcell inhibitory amount of INPROL and/or an opiate compound,

e) transplanting into said mammal the hematopoietic cells of step d

f) optionally treating said mammal in vivo with a stem cell inhibitoryor stimulatory quantity INPROL and/or an opiate compound.

The invention also includes a method of conducting ex vivo stem cellexpansion comprising treating said hematopoietic cells with stem cellinhibitory amounts of INPROL and at least one stimulatory cytokine.INPROL is contacted with the hematopoietic cells before, during and/orafter contact with the stimulatory cytokine. Ex vivo stem cell expansionallows the production of sufficient amounts of stem cells from limitingsources such as cord blood, fetal liver, autologous bone marrow afterchemotherapy, etc. or after purification (e.g., through fluorescentactivated cell sorting using markers such as CD34, CD38 or rhodamine).The ability to selectively grow particular hematopoietic lineages alsoallows the clinician to specifically design stem cell transplantsaccording to the needs of an individual patient.

The invention also includes a method of conducting ex vivo stem cellexpansion comprising treating hematopoietic cells with stem cellstimulatory amounts of INPROL with or without at least one additionalstimulatory cytokine. INPROL is contacted with the hematopoietic cellsbefore, during and/or after contact with the stimulatory cytokine(s). Exvivo, a stem cell stimulator will allow for expansion of stem cellsand/or progenitors while a stem cell inhibitor will maintain stem cellsin their undifferentiated state. The procedure can also be opdmized bythe use of INPROL at stem cell inhibitory amounts in vivo to maintainstem cells in a quiescent state until they are engrafted, after whichINPROL at stem cell stimulatory amounts can be used to stimulate bonemarrow regeneration. Optionally, the hematopoietic cells may be splitinto two preparations and one treated with stem cell stimulatory amountsof INPROL to promote expansion of stem cells and/or progenitors whilethe other is treated with stem cell inhibitory amounts of INPROL tomaintain stem cells in their undifferentiated state. The twopreparations can then be combined and infused into a patient.

The invention also includes a pharmaceutical composition comprising (a)INPROL and (b) at least one inhibitory compound selected from the groupconsisting of MIP-1α, TGFβ, TNFα, INFα, INFβ, INFβ, the pentapeptidepyroGlu-Glu-Asp-Cys-Lys, the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro, andthe tripeptide glutathione (Gly-Cys-γGlu).

The invention also includes a pharmaceutical composition comprising (a)INPROL and (b) at least one stimulatory cytokine selected from the groupconsisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11,IL-13, EL-14, EL-15, G-CSF, GM-CSF, M-CSF, erythropoietin,thrombopoietin, stem cell factor, delta-like protein and flk2/flt3ligand.

The current invention describes an inhibitor of stem cells (INPROL)which is different from those known in the art such as MIP-1α, TGFβ, thetetrapeptide of Frindel and colleagues or the pentapeptide of Paukovitsand coworkers (cf., Wright & Pragnell, 1992 (op. cit.)). Naturallyoccuring native INPROL has a molecular weight exceeding 10,000 daltonsby ultrafiltration which distinguishes it from the tetrapeptide as wellas the pentapeptide. It is more hydrophobic than MIP-1α or TGFβ inreverse phase chromatography systems, distinguishing it from thosecytokines. Further, its mode of action is different from that of anypreviously described inhibitor in that it is active in an in vitro assaywhen used during a preincubation period only. MIP-1α for example, is noteffective when used during a preincubation period only (Example 5).Further, naturally occuring INPROL is active in an assay measuring “highproliferative potential cells” (HPP-PFC) whereas MIP-1α is not (Example6). INPROL is different from those stimulators known in the art such asIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, M-CSF, erythropoietin,thrombopoietin, stem cell factor, and flk2/flt3 ligand. Naturallyoccuring INPROL has little or no sequence similarity to these cytokines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 show an SDS polyacrylamide gel run of the product after eachstage of purification.

FIG. 1—Lane 1 is chymotrypsinogen, Lane 2 is ovalbumnin, Lane 3 is BSA,Lane 4 is fractions <30 kD, Lane 5 is fractions 30-50 kD and Lane 6 isfractions 50-100 kD.

FIG. 2—Lane 1 is after ammonium sulfate precipitation (40-80%) and lanes2-5 are DEAE fractions (Lane #2 represents the active fraction).

FIG. 3—Lane 1 is the supernatant after ammonium sulfate precipitation,Lane 2 is the active DEAE fraction, Lanes 3-5 represent gel filtrationfractions (lane #5 represents the active fraction)

FIG. 4—Lane 2 represents the final product

FIG. 5 shows a reverse phase HPLC chromatogram of the finalpurification.

FIG. 6 shows tritiated thymidine incorporation (cpm) into cells of theFDCP-mix line without (Control=0% Inhibition) and with various amountsof INPROL purified from porcine bone marrow (pINPROL). Data arenormalized against the control value.

FIG. 7 shows the percent of cells in the S phase of the cell cycle aftertreatment of mice with testosterone propionate (TSP), TSP plus pINPROL,or vehicle (Control). Each group contained 25 animals (3-4 per timepoint).

FIG. 8 shows survival of mice treated with two doses of 5-FU, with orwithout pINPROL treatment. Each group contained 30 animals.

FIG. 9 shows survival of irradiated mice, with and without pINPROLtreatment. Each group contained 50 animals.

FIGS. 10A and 10B show regeneration of normal bone marrow long termerculture cells 1 week (10A) and 3 weeks (10B) after treatment with Ara-Cor Ara-C plus pINPROL.

FIG. 11 shows survival of mice (75 per group) after lethal irradiationand transplantation of 3×10⁴ bone marrow cells after pre-incubation withmedium (Control) or pINPROL (25 ng/ml) for 4 hours. Survival wasmonitored for 30 days.

FIG. 12 shows the CFU-GM number formed after 14 days in culture by bonemarrow cells from mice after lethal irradiation and restoration withdonor bone marrow cells preincubated with pINPROL or medium for 4 hours.

FIG. 13 shows suspension cells from lymphoid long-term culture whichwere taken every week, washed out, and plated with IL-7 (10 ng/ml) afterpreincubation with medium or pINPROL for 4 hours.

FIG. 14 shows improved repopulating ability of leukemic peripheral bloodcells treated with pINPROL. Long term culture initiating cells (LTC-IC)were measured by plating adherent and nonadherent LTC cells with andwithout pINPROL, and scoring CFU-GM on day 7. Data are normalized tocontrol values.

FIG. 15A shows a C4 reverse phase chromatogram of purified pINPROLeluting at 53% acetonitrile. Lane 1 is the crude material, Lane 2 ismolecular weight markers and Lane 3 is the purified material.

FIG. 15B shows a C4 reverse phase chromatogram of MIP-1α eluting at43.9% acetonitrile.

FIG. 15C shows an SDS-PAGE chromatogram of the crude pINPROL preparationand of the purified preparation after reverse phase.

FIG. 16 shows hemoglobin sequences:

FIG. 16A shows the cDNA (Seq ID NO:15) and amino acid (Seq ID NO:16)sequences of human alpha hemoglobin and

FIG. 16B shows the cDNA and amino acid (Seq ID NO:17) sequences of humanbeta hemoglobin. Numbering is according to the amino acid.

FIG. 16C shows an amino acid (Seq ID NO:18) sequence comparison of thealpha and beta chains of human (Seq ID NO:16 and Seq ID NO:18), murine(Seq ID NO:19 and Seq ID NO:20) and porcine (Seq ID NO:22) hemoglobins.

FIG. 17 shows a comparison of the C₄ reverse-phase HPLC traces ofpINPROL (FIG. 17A) and of crystallized pig hemoglobin (FIG. 17B).

FIG. 18 shows an SDS-PAGE gel of fractions from a C₄ reverse phase HPLCseparation of crystallized pig hemoglobin. Lane 1 shows molecular weightmarkers, Lane 2 shows Fractions 48-49, derived from the first peak (at47.11 min), Lane 3 shows fractions 50-51, derived from the second peak(at 49.153 min), Lane 4 shows fractions 54-55, derived from the thirdpeak (at 52.25 min) and Lane 5 shows fractions 56-57, derived from thefourth peak (at 53.613 minutes).

FIG. 19 shows a comparison of the 2-dimensional gel electrophoreses ofpINROL (FIG. 19A) and of purified pig beta hemoglobin (FIG. 19B).

FIG. 20 shows a comparison of the effects of purified pig alphahemoglobin, beta hemoglobin or pINPROL in the FDCP-MIX assay.

FIG. 21 shows the reverse phase separation of porcine hemoglobin using ashallow elution gradient.

FIG. 22A shows the plasmid from Hochuli et al., (1988);

FIG. 22B shows the plasmid of Loetscher et al., (1991);

FIG. 22C shows the pDSUb plasmid.

FIG. 23 shows the results of treatment with INPROL on the cobblestoneassay.

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. Thisdescription, while exemplary of the present invention, is not to beconstrued as specifically limiting the invention and such variationswhich would be within the purview of one skilled in this art are to beconsidered to fall within the scope of this invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

INPROL reversibly inhibits or stimulates division of stem cells. Whilenot wishing to be bound to a specific theory, stem cell inhibitors andstimulators are thought to exert their effects by influencing the rateat which stem cells transit through the cell cycle. Specifically, UTROLis effective in temporarily inhibiting or stimulating cell division ofhematopoietic stem cells depending on the amount used. The ability touse a compound clinically which can inhibit or stimulate stem cellproliferation allows for exquisite control of the cycling ofhematopoietic stem cells during, for example, chemotherapy, stem celltransplantation or gene therapy protocols. Thus, the method of thisinvention can be employed in alleviating the undesirable side effects ofchemotherapy on the patient's hematopoietic, myeloid and immune systemsby protecting stem cells from damage caused by chemotherapeutic agentsor radiation used to destroy cancer or virally infected cells or bystimulating recovery after such damage. In one embodiment of theinvention, INPROL is administered to the patient in a dosage sufficientto inhibit stem cell division while the chemotherapeutic agent acts ondiseased cells. After the chemotherapeutic agent has performed itsfunction, the stem cells inhibited by INPROL will, without furthertreatment, revert to dividing cells. If it is desired to enhance theregeneration of hematopoiesis, stimulatory growth factors, cytokines orstem cell stimulatory amounts of INPROL can be used in addition.

As used herein, the term “INPROL” includes mammalian and non-mammalianproteins, purified as in the Examples, hemoglobin, the alpha chain ofhemoglobin (with or without the heme group), the beta chain ofhemoglobin (with or without the heme group), mixtures of alpha and betachains (with or without the heme group), and fragments or analogs ofthese proteins including embryonic, fetal or adult forms (e.g., alpha,beta, gamma, delta, epsilon or zeta chains, either alone or as mixtures,dimers or multimers, with or without the heme group) having the abilityto inhibit and/or stimulate stem cell proliferation. The term “INPROL”includes naturally occurring as well as non-naturally occurring (e.g.,recombinantly and/or synthetically produced) forms of these proteins.The term “INPROL polypeptide” refers to INPROL consisting of 40 or moreamino acids.

As used herein, the term “opiate compounds” are compounds, includingopiates but not INPROL, which bind to opiate receptors (or to receptorsbearing sequence relatedness to opiate receptors, e.g., ORL1) and exerteither agonist, antagonist or mixed agonist/antagonist activities. Forexample, specific agonists and antagonists exist for mu receptors (whichare selectively activated by DAMGO and DALDA and selectively antagonizedby CTOP and naloxonazine), for kappa receptors (which are selectivelyactivated by GR 89696 fumarate or U-69593 and selectively antagonized bynor—binaltorphimine hydrochloride) and for delta receptors (which areselectively activated by DADLE and DPDPE and selectively antagonized bynatrindole). In addition, there are broad-spectrum antagonists (such asnaloxone) and agonists (such as etorphine) which act on all threereceptor subtypes. Nociceptin specifically agonizes the ORL1 receptor.Opiate compounds with stem cell stimulatory and/or inhibitory activitiescan be used for each of the applications described herein for INPROL.

As used herein, “stem cell stimulatory amount” is that amount whichinduces proliferation of stem cells. As used herein, “stem cellinhibitory amount” is that amount which inhibits proliferation of stemcells. In all cases, both in vivo and ex vivo, the amount selected willdepend upon the specific INPROL or opiate compound selected and thespecific condition or application; in particular, equimolar doses ofpolypeptides or fragments of INPROL are active as are equimolar opiatepeptides or small molecules.

In a typical clinical situation, where stem cell inhibition is desired,INPROL is administered to a patient in a daily regimen by intravenousinjection or infusion in dosage unit form using, for example, 0.01 to100 mg/kg, advantageously 0.1 to 1.0 mg/kg, of INPROL administered,e.g., 4 to 60 hours prior to standard chemotherapy or radiationtreatments when it is desirable to inhibit stem cell cycling.

In situations where stimulation of stem cell cycling is desirable, suchas to promote recovery after chemotherapy or radiation, INPROL at stemcell stimulatory amounts are used. Such doses are typically 1-500 mg/kg,advantageously 10 mg to 100 mg/kg.

In cases where it is desirable to use opiate compound(s) to inhibit orto stimulate stem cell cycling, the opiate compound(s) are used atequimolar concentrations to that described for INPROL.

In another embodiment of the invention, pretreatment with INPROL at stemcell inhibitory amounts allows for increased doses of chemotherapeuticagents or of radiation beyond doses normally tolerated in patients.Similarly, post-chemotherapy or post-radiation treatment with INPROL atstem cell stimulatory amounts also allows for increases in normallytolerated doses of chemotherapy or radiation.

A large fraction of hematopoietic stem cells are normally quiescent(slowly or non-cycling). However, as a compensatory response tochemotherapy-induced hematopoietic damage, a larger proportion of stemcells enter into cycling after chemotherapy, which makes themparticularly vulnerable to subsequent doses of cytotoxic chemotherapy ortherapeutic irradiation. By inhibiting cycling of such stem cells,INPROL treatment permits earlier or more frequent administration ofsubsequent doses of cytotoxic chemotherapy, either at conventional orelevated doses.

Some normal individuals have stem cells that spontaneously cyclerapidly; INPROL at stem cell inhibitory amounts is useful in suchindividuals even if given prior to the first dose of radiation orchemotherapy.

In one embodiment of the invention, INPROL (0.1 mgs, to 6 gms/kg bodyweight—advantageously 1.0 to 60 mgs./kg) is administered about 24 hoursto 10 days after an initial dose of chemotherapy. After another 4 to 60hours, advantageously 24 to 48 hours, another dose of chemotherapy isadministered. This cycle of alternating chemotherapy and INPROL iscontinued according to therapeutic benefit. Chemotherapy agents andprotocols for administration are selected according to suitability forparticular tumor types in standard clinical practice. Optionally,stimulatory growth factors such as G-CSF, stem cell factor, or INPROL atstem cell stimulatory amounts is used after chemotherapy or radiationtreatment to further improve hematopoietic reconstitution.

For ex vivo applications 0.1 ng to 100 ng/10⁶ cells/ml, advantageously2-50 ng/10⁶ cells/ml, of INPROL are used in cases where inhibition ofstem cell proliferation is desired. For cases where stem cellstimulation is desired, 10 ng-100 μg/10⁶ cells/ml, advantageously 1-100μg/10⁶ cells/ml, of INPROL are used.

In cases where it is desirable to use opiate compound(s)to inhibit orstimulate stem cell cycling, the opiate compound(s) are used atequimolar concentrations to that described for INPROL.

In another embodiment of the invention, INPROL is employed in a methodfor preparing autologous hematopoietic cells for transplantation. Thehematopoietic cells are treated ex vivo with an effective amount ofINPROL to inhibit stem cell division and then purged of cancerous cellsby administering to the marrow cultures an effective amount of achemotherapeutic agent or radiation. Chemotherapy agents withspecificity for cycling cells are preferred. Marrow thus treated isreinjected into the autologous donor. Optionally, the patient is treatedwith stem cell stimulatory amounts of INPROL and/or another agent knownto stimulate hematopoiesis to improve the hematopoietic reconstitutionof the patient. Such a technique allows for effective purging of tumorcells during autologous bone marrow grafts while protectinghematopoietic stem cells. Such protection can be afforded with either exvivo or in vivo purging protocols. Once successfully transplanted, thereis a need for stem cells to rapidly proliferate to regenerate normalbone marrow function. This can be afforded by the use of INPROL at stemcell stimulatory amounts which stimulates cycling of stem cells andenhances recovery of bone marrow function.

In another embodiment of the invention, INPROL is employed in a methodfor preparing hematopoietic cells for gene therapy. The hematopoieticcells are treated ex vivo with INPROL at stem cell stimulatory amountsand/or other stimulatory cytokine(s) to stimulate stem cell division,and then transfected (advantageously infected using e.g. a retroviralvector) with the gene(s) of interest. After transfection has beenachieved, cells are washed and treated with INPROL at stem cellinhibitory amounts to return stem cells to quiescence. Marrow thustreated is reinjected into the donor. Optionally, the patient is treatedin vivo with INPROL at stem cell inhibitory amounts to maintain stemcells in their quiescent form and to increase their marrow repopulatingability.

In another embodiment of the invention, INPROL is employed as anadjunctive therapy in the treatment of leukemia. For example, in diseasestates where the leukemic cells do not respond to INPROL, thehematopoietic cells are treated ex vivo with INPROL at stem cellinhibitory amounts. The proliferation of normal stem cells is preventedby administration of INPROL. Thus, during the time that theproliferating leukemic cells are treated with a cell cycle-specificcytotoxic agent, a population of normal stem cells is protected fromdamage. Additionally, a stimulatory cytokine, such as IL-3, GM-CSF, isoptionally administered to induce cycling in the leukemic cells duringdrug or radiation treatment while the normal stem cells are protectedwith INPROL. The patient is treated with chemotherapy agents orradiation to destroy leukemic cells, and the purged marrow is thentransplanted back into the patient to establish hematopoieticreconstitution.

Similarly, in another embodiment of the invention for treatment ofpatients with serious viral infections that involve blood cells orlymphocytes, such as HIV infection, hematopoietic cells are treated exvivo or in vivo with INPROL followed by antiviral agents, drugs whichdestroy infected cells, or antibody-based systems for removing infectedcells. Following myeloablative antiviral or myeloablative chemotherapyto eradicate viral host cells from the patient, the INPROL-treatedmarrow cells are returned to the patient.

In another embodiment of the invention, INPROL is employed to treatdisorders related to hyperproliferative stem cells. For example,psoriasis is a disorder caused by hyperproliferating epithelial cells ofthe skin and is sometimes treated with cytotoxic drugs. Otherpre-neoplastic lesions in which stem cell proliferation is involved arealso amenable to effective amounts of INPROL employed to inhibit theproliferation of the stem cells. Patients with acquired immunedeficiency syndrome have abnormally high rates of stem cell cyclingresulting in stem cell exhaustion; these patients also benefit fromtreatment with effective amounts of INPROL to inhibit stem cell cycling.For these uses, topical or transdermal delivery compositions (e.g.,ointments, lotions, gels or patches) containing INPROL are employedwhere appropriate, as an alternative to parenteral administration.

In most cases of leukemia, the leukemia progenitors are differentiatedcell populations which are not affected by INPROL and which aretherefore treated by methods using INPROL such as those described above.In cases where leukemia progenitors are very primitive and are directlysensitive to inhibition by INPROL, proliferation of leukemia cells isattenuated by administration of effective amounts of INPROL.

In another embodiment of the invention, INPROL is employed to treatdisorders related to hypoproliferative stem cells. For example,myelodysplasic syndromes and aplastic anemia are disorders caused byhypoproliferating stem cells of the bone marrow. Other syndromes inwhich stem cell hypoproliferation is involved are treatable with stemcell stimulating amounts of INPROL.

Antibodies, monoclonal or polyclonal, are developed by standardtechniques to the INPROL peptides or polypeptides. These antibodies orINPROL peptides or polypeptides are labeled with detectable labels ofwhich many types are known in the art. The labeled INPROL or anti-INPROLantibodies are then employed as stem cell markers to identify andisolate stem cells by administering them to a patient directly fordiagnostic purposes. Alternatively, these labeled peptides, polypeptidesor antibodies are employed ex vivo to identify stem cells in ahematopoietic cell preparation to enable their removal prior to purgingneoplastic cells in the marrow. In a similar manner, such labeledpeptides, polypeptides or antibodies are employed to isolate andidentify epithellal or other stem cells. In addition, such antibodies,labeled or unlabeled, are used therapeutically through neutralization ofINPROL activity or diagnostically through detection of circulatingINPROL levels.

INPROL can be cloned from human gene or cDNA libraries for expression ofrecombinant human INPROL using standard techniques. For example, usingsequence information obtained from the purified protein, oligonucleotideprobes are constructed which can be labeled, e.g., with 32-phosphorus,and used to screen an appropriate cDNA library (e.g., from bone marrow).Alternatively, an expression library from an appropriate source (e.g.,bone marrow) is screened for cDNA's coding for INPROL using antibody orusing an appropriate functional assay (e.g., that described in Example2). Hemoglobin itself, as well as the individual alpha and beta chains,have been cloned and expressed using methods known in the state of theart (see Pagnier et al., Rev. Fr. Transfus. Hemobiol. 35:407-15, 1992;Looker et al., Nature 356:258-60, 1992; Methods in Enzymology vol. 231,1994).

The present invention includes DNA sequences which include: theincorporation of codons “preferred” for expression by selectednonmammalian hosts: the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences which facilitate construction ofreadily-expressed vectors or production or purification of the alpha,beta, gamma, delta, epsilon and/or zeta chain of hemoglobin.

The present invention also provides DNA sequences coding for polypeptideanalogs or derivatives of hemoglobin alpha, beta, gamma, delta, epsilonand/or zeta chains which differ from naturally-occurring forms in termsof the identity or location of one or more amino acid residues (i.e.,deletion analogs containing less than all of the residues specified;substitution analogs, wherein one or more residues specified arereplaced by other residues; and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptide) and which share some or all of the properties ofnaturally-occurring forms.

In an advantageous embodiment, INPROL is the product of prokaryotic oreukaryotic host expression (e.g., by bacterial, yeast, higher plant,insect and mammalian cells in culture) of exogenous DNA sequencesobtained by genomic or cDNA cloning or by gene synthesis. That is, in anadvantageous embodiment, INPROL is “recombinant INPROL”. The product ofexpression in typical yeast (e.g., Saccharomyces, cerevisiae) orprokaryote (e.g., E. coli) host cells are free of association with anymammmlian proteins. The products of expression in vertebrate(e.g.,non-human mammalian (e.g., COS or CHO) and avian) cells are freeof association with any human proteins. Depending upon the hostemployed, polypeptides of the invention can be glycosylated or can benon-glycosylated. Polypeptides of the invention optionally also includean initial methionine amino acid residue (at position −1).

The present invention also embraces other products such as polypeptideanalogs of the alpha, beta, gamma, delta, epsilon and/or zeta chain ofhemoglobin. Such analogs include fragments of the alpha, beta, gamma,delta, epsilon and/or zeta chain of hemoglobin. Following well knownprocedures, one can readily design and manufacture genes coding formicrobial expression of polypeptides having primary sequences whichdiffer from that herein specified for in terms of the identity orlocation of one or more residues (e.g., substitutions, terminal andintermediate additions and deletions). Alternatively, modifications ofcDNA and genomic genes can be readily accomplished by well-knownsiteirected mutagenesis techniques and employed to generate analogs andderivatives of the alpha, beta, gamma, delta, epsilon or zeta chains ofhemoglobin. Such products share at least one of the biologicalproperties of INPROL but can differ in others. As examples, products ofthe invention include the alpha, beta, gamma, delta, epsilon or zetachains which is foreshortened by e.g., deletions; or those which aremore stable to hydrolysis (and, therefore, can have more pronounced orlonger-lasting effects than naturally-occurring); or which have beenaltered to delete or to add one or more potential sites forO-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by, e.g., alanine or serineresidues and are more easily isolated in active form from microbialsystems; or which have one or more tyrosine residues replaced byphenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are peptide or polypeptidefragments duplicating only a part of the continuous amino acid sequenceor secondary conformations within the alpha, beta, gamma, delta, epsilonor zeta chains which fragments can possess one property of INPROL (e.g.,receptor binding) and not others (e.g., stem cell inhibitory activity).It is noteworthy that activity is not necessary for any one or more ofthe products of the invention to have therapeutic utility (see, Weilandet al., Blut 44:173-5, 1982) or utility in other contexts, such as inassays of inhibitory factor antagonism. Competitive antagonists areuseful in cases of overproduction of stem cell inhibitors or itsreceptor.

In addition, peptides derived from the protein sequence which retainbiological activity can be chemically synthesized using standardmethods. The present invention also provides for sequences coding forpeptide analogs or derivatives of hemoglobin alpha, beta, gamma, delta,epsilon and/or zeta chain which differ from naturally-occurning forms interms of the identity or location of one or more amino acid residues(e.g., deletion analogs containing less than all of the residuesspecified; substitution analogs, wherein one or more residues specifiedare replaced by other residues, either naturally occuring or otheranalogs known in the state of the art such as D-amino acids; andaddition analogs wherein one or more amino acid residues is chemicallymodified to increase stability, solubility and/or resistance toproteolysis) and which share some or all of the properties ofnaturally-occurring forms.

Peptide sequences such as described above can be identified by a varietyof means. Comparison of the three dimensional structures of nativehemoglobin chains active in the assay (e.g., alpha chain) withstructurally related proteins which are inactive (e.g., myoglobin) canidentify regions which have different conformations in three-dimensionalspace and which are therefore candidate regions for active peptides.Another approach uses selective proteolysis, in which proteolyticenzymes are used in limited digests of hemoglobin chains resulting inpeptides which can separated, for example, by reverse phase HPLC andthen assayed for stem cell inhibition. Peptides can also be generated bychemical synthesis (e.g., solid phase synthesis); a series ofoverlapping peptides (e.g., 15-mers) which encompass the sequence of thehemoglobin chain of interest (e.g., alpha chain) can easily be generatedand tested in stem cell assays. Combinatorial libraries can be generatedin which multiple chemical syntheses are conducted and where selectedamino acid positions are made variable resulting in large numbers ofpeptide analogs for screening (e.g., Dooley et al., Peptide Research8:124-137, 1995). Alternatively, recombinant methods can be employed.Site directed mutagenesis can be used to identify critical residuesnecessary for activity of a particular hemoglobin chain. Regions of achain which is known to be active as a stem cell inhibitor (e.g., alphachain) can be substituted with regions from a related but inactiveprotein (e.g., myoglobin) and tested in stem cell assays, allowing foridentification of regions necessary for activity. Such identifiedregions can be expressed as peptides and tested for activity in stemcell cycling assays.

Homologous or analogous versions of INPROL from other species areemployed in various veterinary uses, similar to the therapeuticembodiments of the invention described above.

INPROL at stem cell inhibitory amounts act on cycling stem cells byreversibly placing them in an undividing or slowly dividing “resting”state. When it is desirable to stimulate the quiescent stem cells intodivision, e.g., after treatment of a patient with cancer chemotherapyagents or radiation, INPROL at stem cell stimulatory amounts can beused. Alternatively, or in addition, colony-stimulating factors andother hematopoietic stimulants are administered to the subject Examplesof such factors include but are not limited to: M-CSF (CSF-1), GM-CSF,G-CSF, Megakaryocyte-CSF, thrombopoieitin, stem cell factor or othercytokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9,IL-11, IL-12, IL-13, IL-14, or erythropoietin .

INPROL polypeptides or active fragments having stem cell inhibitoryactivity are purified or synthesized by conventional chemical processescombined with appropriate bioassays for stem cell inhibitory activity,as exemplified in the protocols described below.

In one embodiment of the invention, a therapeutically effective amountof the INPROL protein or a therapeutically effective fragment thereof isemployed in admixture with a pharmaceutically acceptable carrier. ThisINPROL composition is generally administered by parenteral injection orinfusion. Subcutaneous, intravenous, or intramuscular injection routesare selected according to therapeutic effect achieved.

When systemically administered, the therapeutic composition for use inthis invention is in the form of a pyrogen-free, parenterally acceptableaqueous solution. Pharmaceutically acceptable sterile protein solution,having due regard to pH, isotonicity, stability, carrier proteins andthe like, is within the skill of the art.

Also comprehended by the invention are pharmaceutical compositionscomprising therapeutically effective amounts of peptide or polypeptideproducts of the invention together with suitable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriersuseful in INPROL therapy. A “therapeutically effective amount” as usedherein refers to that amount which provides a therapeutic effect for agiven condition and administration regimen. Such compositions areliquids, gels, ointments, or lyophilized or otherwise dried formulationsand include diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength, additives such as albumin or gelatinto prevent adsorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), solubihzing agents (e.g., glycerol,polyethylene glycol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol,parabens), bulking substances or tonicity modifiers (e.g., lactose,mannitol), covalent. attachment of polymers such as polyethylene glycolto the protein, complexation with metal ions, or incorporation of thematerial into or onto particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, hydrogels, etc. or intoliposomes, niosomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, biodegradable injectable microcapsules ormicrospheres, or protein matrices, erythrocyte ghosts, spheroplasts,skin patches, or other known methods of releasing or packagingpharmaceuticals. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of INPROL. Controlled or sustained release compositionsinclude formulation in lipophilic depots (e.g., fatty acids, waxes,oils). Also comprehended by the invention are particulate compositionscoated with polymers (e.g., poloxamers or poloxamines) and INPROLcoupled to antibodies directed against tissue-specific receptors,ligands or antigens or coupled to ligands of tissue-specific receptors.Other embodiments of the compositions of the invention incorporateparticulate forms of protective coatings, protease inhibitory factors orpermeation enhancers for various routes of administration, includingparenteral, pulmonary, nasal, topical (skin or mucosal) and oral. Inanother embodiment, the composition containing INPROL is administeredtopically or through a transdermal patch.

In one embodiment, the compositions of the subject invention arepackaged in sterile vials or ampoules in dosage unit form.

The invention also comprises compositions including one or moreadditional factors such as chemotherapeutic agents (e.g., 5-fluorouracil(5FU), cytosine arabinoside, cyclophosphamide, cisplatin, carboplatin,doxyrubicin, etoposide, taxol, alkylating agents), antiviral agents(e.g., AZT, acyclovir), TNF, cytokines (e.g., interleukins),antiproliferative drugs, antimetabolites, and drugs which interfere withDNA metabolism.

The dosage regimen involved in a method for treating the subjectanticipating exposure to such cytotoxic agents or for treatment ofhyperproliferating stem cells is determined by the attending physicianconsidering various factors which modify the action of drugs; e.g., thecondition, body weight, sex, and diet of the patient, the severity ofany infection, time of administration and other clinical factors.

Following the subject's exposure to the cytotoxic agent or radiation,the therapeutic method of the present invention optionally employsadministering to the subject INPROL at stem cell stimulatory amountsoptionally including one or more lymphokines, colony stimulating factorsor other cytokines, hematopoietins, interleukins, or growth factors togenerally stimulate the growth and division of the stem cells (and theirdescendants) inhibited by the prior treatment with INPROL. Suchtherapeutic agents which encourage hematopoiesis include IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, Meg-CSF, M-CSF (CSF-1), GM-CSF, G-CSF orerythropoietin. The dosages of these agents are selected according toknowledge obtained in their use in clinical trials for efficacy inpromoting hematopoietic reconstitution after chemotherapy orhematopoietic stem cell transplant. These dosages would be adjusted tocompensate for variations in the physical condition of the patient, andthe amount and type of chemotherapeutic agent or radiation to which thesubject was exposed. Progress of the reversal of the inhibition of thestem cells caused by administration of INPROL in the treated patient ismonitored by conventional methods.

In the treatment of leukemia, it is beneficial to administer both INPROLto inhibit normal stem cell cycling and a stimulator of leukemic cellgrowth, such as IL-3 or GM-CSF, simultaneously with the cytotoxic drugtreatment or during irradiation. By this protocol, it is possible toachieve the greatest differences between the cycling statuses and drugsensitivities of normal and leukemic cells.

EXAMPLE 1 In Vivo Stem Cell Proliferation Inhibition Assay

For the detection of stem cells proliferation the number of CFU-S inS-phase of the cell cycle was measured by the ³H-Thymidine “suicide”method (Becker et al., Blood 26:296-308, 1965).

Immature hematopoietic progenitors-Colony Forming Units in spleen(CFU-S)—can be detected in vivo by forming macroscopic colonies in thespleens of lethally irradiated mice, 8-12 days after the intravenousinjection of hematopoietic cells (Till & McCulloch, 1961).

For the standard CFU-S proliferation assay the method of ³H-Thymidine“suicide” is usually applied (Becker et al., 1965). The method is basedon incorporation of radiolabelled Thymidine, (³H-Thymidine) a precursorof DNA into cells during DNA synthesis. The CFU-S which are in S-phaseof the cycle at the time of testing, are killed by the highradioactivity and therefore not able to form colonies in spleen. Thus,the difference between the number of CFU-S formed by the injection ofthe cell sample incubated without ³H-Thymidine and the same cellsincubated with ³H-Thymidine shows the percentage of the proliferatingCFU-S in the original sample.

The inhibitor testing can not be done with the bone marrow stem cellpopulation from unstimulated animals, as far as the inhibitor onlyeffects cycling CFU-S, which are as low as 7-10% of the total CFU-Spopulation in the bone marrow of normal mice.

To stimulate CFU-S proliferation, phenylhydrazine (PHZ), or sublethalirradiation were used (Lord, 1976).

We have developed the method of using testosterone-propionate (TSP)based on its stimulatory effect on CFU-S cycling (Byron et al., Nature228:1204, 1970) which simplified the testing and did not cause any sideeffects. The TSP induced stimulation of CFU-S proliferation within 20-24hours after injection and the effect could be seen for at least 7 days.

The procedure used for the screening of the fractions duringpurification of the Inhibitor was as follows:

Mice: BDF₁ or CBF₁ mice strains were used throughout all testing.

Donor mice were treated with a 10 mg/100 g dose of TSP byintraperitoneal injection of 0.2 ml/mouse in order to induce 30-35% ofthe CFU-S into S-phase.

Twenty-four hours later the bone marrow is taken from the femurs for thecell suspension preparation. Five to ten million cells per ml are thenincubated with different control and test fractions for 3.5 hours at 37°C. in water bath, with two tubes for each group (one for hot(radioactive) and one for cold (non-radioactive)).

After 3.5 hours, ³H-Thymidine (1 mCi/ml, specific activity 18-25Ci/mmole) is added to each hot tube in a volume of 200 μl per 1 ml ofcell suspension; nothing is added to the cold tubes. Incubation iscontinued for 30 more minutes at 37° C.

After the 30 minute incubation, the kill reaction is terminated byadding 10 ml of cold (4° C.) medium containing 400 μg/ml nonradioactiveThymidine. Cells are washed extensively (3 times).

Cells are resuspended and diluted to a desirable concentration for theinjections, usually 2-4×10⁴ cells per mouse in 0.3-0.5 ml.

Recipient mice, 8-10 per group, are irradiated not later than 6 hoursbefore the injections.

Recipient spleens are harvested on day 9-12 and fixed in Tellesnitsky'ssolution; the colonies are scored by eye score. The percentage of cellsin S-phase are calculated using the formula.${\% \quad S} = {\frac{a - b}{a} \times \left( {100\%} \right)}$

where a—CFU-S number without ³H-Thymidine

where b—CFU-S number with ³H-Thymidine

The test data of INPROL presented in Table 1 demonstrate that cyclingstem cells after treatment with INPROL become resistant to the action of³H-Thymidine. For this and all of the following examples, the term“pINPROL” refers to the purified protein from porcine bone marrow. Thesame protection is seen for the S-phase specific cytotoxic drugscytosine arabinoside and hydroxyurea (data not shown). If the treatedstem cells are then washed with the cold media containingnon-radioactive Thymnidine, the surviving stem cells proliferate inmouse spleens to form colonies normally.

TABLE 1 Inhibitory Activity Of pINPROL On CFU-S Proliferation DuringFour Hour Incubation With Bone Marrow Cells Percent CFU-S Group −³H-TdR+³H-TdR Killed by ³H-TdR No incubation 22.2 ± 2.0* 13.7 ± 2.4* 38.3 ±1.7 4 Hour with 18.7 ± 3.0* 11.4 ± 1.3* 43.1 ± 1.4 Media 4 Hour with21.2 ± 2.3* 20.7 ± 2.6*  2.1 ± 0.08 pINPROL *CFU-S per 2 × 10⁴ cells

EXAMPLE 2 In Vitro Stem Cell Proliferation Inhibition Assay

Using the following test system (Lord et al., in The Inhibitors ofHematopoiesis pp. 227-239, 1987) the direct effect of INPROL was shown.The multilineage factor (IL-3) dependent stem cell line, FDCP mix A4(A4), was maintained in IMDM medium supplemented with 20% horse serumand 10% WEHI-3-conditioned medium as a source of colony-stimulatingIL-3.

A tritiated Thymidine incorporation assay was used to measureproliferation: A4 cells (5×10⁴ in 100 μl medium with 20% horse serum and50% of WEHI-3 CM) were incubated at 37° C. in 5% CO₂ for 16 hours.

pINPROL or the crude BME (fraction IV) were added at the start.Tritiated thymidine ((³H-Tdr) 3.7 KBq in 50 μl at 740 GBq/mmole) wasthen added to each group for a further 3 hours of incubation. The levelof proliferation was measured by harvesting cells and the % inhibitoncalculated using the formula

% Inhibition=cpm without INPROL−cpm with INPROL×(100%) cpm withoutINPROL

Incorporation of tritiated thymidine (³H-Tdr) by FDCPmix-A4 cells grownin the presence of graded doses of normal bone marrow extract or pINPROLis depicted on FIG. 6. It can be seen that purified composition ofpINPROL is at least 1,000 times more active than the starting material.Time of exposure (16 hours) is an important factor for effectiveinhibition and shows the evidence of the direct effect of pINPROL onstem cells of the A4 cell line.

EXAMPLE 3 Inhibition of CFU-S Proliferation by INPROL Injected in vivoDoses and the Duration of the Effect

The studies of the effect of INPROL injected in vivo revealed thatINPROL can effectively block the recruitment of CFU-S into cycle, thusprotecting those cells from the cytotoxic effect of further treatment,showing its potential for clinical use.

The experimental protocol had two goals: to check the effect of INPROLon CFU-S when injected in vivo and to define the effective duration ofINPROL activity in relation to cycling stem cells.

To stimulate CFU-S proliferation, the injection oftestosterone-propionate was used based on the effect mentioned above inExample 1.

Mice BDF1 were injected with TSP (10 mg/100 g) on Day 0; 24 hours latermice of each experimental group (4 mice per group) received a singlepINPROL injection at doses of 0 μg, 5 μg, 10 μg, and 15 μg/mouse i.p.

Twenty-four hours after pINPROL injection, mice were sacrificed and thepercent of cycling CFU-S was measured by the assay described inExample 1. TSP injection induced about 50% CFU-S into cycling incomparison with 7% in untreated mice. pINPROL in doses as low as 2μg/mouse was able to inhibit TSP induced proliferation down to thenormal level.

For the duration of the effect evaluation, one group of mice (21 miceper group) was injected with TSP only and another group was injectedboth with TSP and pINPROL (24 hours after TSP). The CFU-S cycling wasmeasured every 24 hours during a week by taking 3 donors from each groupand measuring CFU-S cycle status in their bone marrow by methoddescribed (see Example 1). Data presented in FIG. 7 show that while theduration of the effect of TSP is at least 7 days, a single injection ofINPROL can place CFU-S into quiescence and keep them out of cycle for nomore than 48-72 hours. Since the majority of chemotherapeutic agentsused for cancer and leukemia chemotherapy have a relatively short invivo half-life, usually less than 24 hours, the INPROL effect accordingto the data obtained is maintained for longer than the effective timeduring which the chemotherapeutic agents like cytosine arabinoside orhydroxyurea are active in vivo. More importantly, for chemotherapeuticand radiation treatments having longer intervals (more than 24 hours andless than 96 hours) between the first (non-damaging for the stem cells)and the second (damaging to the CFU-S) treatments, a single injection ofINPROL during the intervals between the two applications ofchemotherapeutic agent or radiation should be sufficient. For severalrepeatable cycles of cytotoxic therapy or radiation the same strategycould be applied based on the duration of the INPROL effect.

EXAMPLE 4 Most Primitive Hematopoietic Stem Cells Stimulated to CycleRapidly After Treatment with 5-FU are Protected by INPROL from theSecond 5-FU Exposure

The drug 5-fluorouracil (5-FU) drastically reduces the number of cellsin the myeloid and lymphoid compartments. It is usually thought of asbeing cell-cycle specific, targeting rapidly proliferating cells,because incorporation of the nucleotide analogue into DNA during S-phaseof the cell cycle or before results in cell death. The long-termsurvival and immunohematopoietic reconstitution of the bone marrow ofmice is not affected by a single dose of 5-FU; however, it wasdemonstrated (Harrison et al. Blood 78:1237-1240, 1991) that pluripotenthematopoietic stem cells (PHSC) become vulnerable to a second dose of5-FU for a brief period about 3-5 days after the initial dose. It can beexplained that PHSC normally cycle too slowly for a single dose of 5-FUto be effective and are stimulated into rapid cycling by stimuliresulting from the initial 5-FU treatment. We have proposed that PHSCcan be returned to a slow cycle status by INPROL and thus protected fromthe second 5-FU treatment.

The mice used in these experiments were BDFI male mice. A stock solutionof 5-FU (Sigma) was prepared in physiologic saline at a concentration of10 μg/ml. Each treated mouse received 2 mg of 5-FU per 10 g body weightvia a tail vein at Day 0 of the experiment; 24 hours later mice wereinjected with pINPROL (10 μg/100 g of body weight) intraperitoneally andon Day 3 were injected, with the second dose of 5-FU. The survival studywas conducted by monitoring the death of mice in experimental (treatmentwith pINPROL) and control groups of 30 mice each. The survival curvesare shown in FIG. 8.

EXAMPLE 5 Effects of Pre-Incubation with INPROL vs. MIP-1α in BoneMarrow Cells

The purpose of this experiment was to compare the inhibitory effects ofpre-incubation with pINPROL and MIP-1α on mouse bone marrow cells invitro.

The following procedure was used:

in vivo: BDF1 mice, 6-15 weeks of age, are injected with 200 mg/kg 5FUi.p. 48 hours before harvesting marrow from the femurs.

in vitro: A single cell pooled suspension is counted and 5×10⁶ cells areincubated in a total of 2 mls with or without pINPROL or MIP-1α, with 5%horse serum, IMDM media with added L-glutamine, at 37° C. and 5% CO₂ for4 hours. The cells are then washed twice and recounted. They are platedin methylcellulose in the following final conditions:

0.8% methylcellulose

125% horse serum

20 ng/ml recombinant murine IL-3

L-glutanine added

5×10⁵ cells per ml

IMDM media

Plates were incubated for 11 days at 37° C. and 5% CO₂ in 100% humidity.Colonies more than 50 cells were counted.

TABLE 2 Groups Colony Number Percent of Control Control 31.0 100%pINPROL 21.25 68.5%  MIP-1α 35.25 114%

EXAMPLE 6 INPROL Inhibits HPP-CFC Proliferation

An in vitro assay for assessing murine reconstituting stem cells andearly precursors is the high proliferative potential colony (HPP-PFC)assay; other related assays, e.g., CFU-A, CFU-GM, CFU-E, and CFU-GEMM,detect progressively restricted progenitor populations (M. Moore, Blood177:2122-2128, 1991). This example shows that pretreatment of cells withpINPROL inhibits their proliferation, whereas MIP-1α fails to do sounder these experimental conditions.

BDF1 mice were treated with 5-fluorouracil (200 mg/kg i.p.) before theirbone marrow was assayed for HPP-CFC numbers. Cells were washed bycentrifugation and incubated at densities of 10⁶ to 5×10⁶/ml in mediumwith either no added agent (Controls), pINPROL (25 ng/ml) or MIP-1α (200ng/ml) for 4 hours. After incubation, cells were washed and plated inagar (0.3%) with 30% FCS and combined conditioned medium from 5637 andWEHI-3B cell lines (7.5% of each conditioned medium, as recommended bySharp et al., 1991). Plating concentration was 5×10⁴ cells/ml in 60 mmdishes. Colonies were scored on day 14 and the results are indicatedbelow.

TABLE 3 Group HPP-CFU % of Control Control 15.5 ± 1.2 100% pINPROL  8.3± 0.7 53.5%  MIP-1α 15.8 ± 0.9 101%

According to these results, MIP-1α did not inhibit proliferation of themost immature precursors when present only during the pre-incubationperiod. pINPROL did effectively inhibit proliferation under theseconditions, indicating fundamental differences between pINPROL andMIP-1α in terms of biological activity.

EXAMPLE 7 INPROL Therapy Effect on the Recovery from Radiation-inducedBone Marrow Aplasia

Bone marrow aplasia is the primary limiting toxicity of radiation cancertherapy. It has been demonstrated that some growth factors (e.g., G-CSF,GM-CSF, erythropoietin) can accelerate recovery from radiation-inducedbone marrow aplasia. The concept of protection by using an inhibitor ofstem cell proliferation is a different and complementary approach incoping with hematological damage. To follow the treatment proceduredeveloped earlier (Examples 3, 4) a model of lethal irradiation of micewas established. It is known in the art that mice receiving 9Gy ofcobalt 60 start dying after 10-14 days; by Day 30, mortalityapproximates 50%. This lethal dose was used in our model by splitting itinto two subsequent applications of 4.5Gy each with an interval 3 daysbetween doses. Preliminary data showed that the survival curve in thatmodel was very close to that known for a single irradiation with 9Gy;moreover the test for the CFU-S proliferation showed that 24 hours afterthe first irradiation, 35-50% of CFU-S are induced to proliferate. Suchcells can be protected by a stem cell inhibitor delivered prior to thesecond dose.

To examine this possibility, mice (50 mice/group) received 4.5Gy on Day0. Twenty four hours later, one group received pINPROL (2 μg/mouse i.p.)and another, control group was injected with saline. The second dose ofradiation (4.5 Gy) was given on Day 3.

FIG. 9 shows the increased survival after a single dose of pINPROL. Theconditions of the model are clinically relevant for treating any cancer,including those characterized by solid tumors; such treatment would beadministered to a patient having cancer by delivering an effective doseof INPROL between two consecutive dosages of radiation, thereby allowinggreater dosages of radiation to be employed for treatment of the cancer.It should also be possible to extend this modality to chemotherapeuticagents.

EXAMPLE 8 INPROL Use for the Autologous Bone Marrow Transplantation

Bone marrow transplantation is the only known curative therapy forseveral leukemias (CML, AML, and others). Ex vivo conditioning ofautologous BMT for infusion should provide potential autologous sourcesof normal stem cells free of leukemic contamination and able torepopulate the recipient's hematopoietic system to allow aggressive andeffective therapy.

1. Long-term Bone Marrow Culture L1210 Leukemia Model for the Study ofINPROL Effect Preserving Normal Hematopoiesis During Pursing with AraC.

Long-Term Bone Marrow Cultures (LTBMC) were established according toToksoz et al. (Blood 55:931-936, 1980) and the leukemic cell line L1210was adopted to the LTBMC by co-cultivation during 2 weeks. Thesimultaneous growth of normal and leukemic progenitors occurred in thesecombined LTBMC/L1210 cultures, similar to the situation in the bonemarrow of a leukemic patient. Discrimination between normal colonyforming units CFU and leukemic CFU was possible by growing them as agarcolonies in the presence or absence of the conditioned medium fromWEHI-3 (a murine IL-3 producing cell line). Normal cells undergoapoptosis in the absence of IL-3 whereas leukemic cells can formcolonies in its absence. Suspension cells from LTBMC-L1210 compositiongive approximately 150 colonies in presence of IL-3 (normalhematopoietic clones) and 70 colonies when growing without IL-3(leukemic clones) per 50,000 cells plated.

The procedure of purging was as follows: on Day 0 all suspension cellsand media (10 ml/flask) were taken off the flasks with LTBMC-L1210 andreplace with 2 ml of media containing 200 μg cytosine arabinoside (AraC)(Tsyrlova et al. in Leukemia: Advances in Biology and Therapy v. 35,1988); after 20 hours of incubation, flasks were washed out and replacedwith 2 ml of fresh media alone (control group) or media containingpINPROL at 25 ng/ml for 4 hours. After this preincubation, cells wereincubated again with 100 μg/flask AraC for 3 hours at 37° C. Each groupcontained 4 flasks. LTBMC-L1210 cultures were washed 3 times andreplaced with fresh LTBC media; they were maintained as before for theregeneration studies for 3-4 weeks.

Data are presented in FIG. 10. There was no cell growth seen in controlcultures treated with AraC only, while in INPROL protected flasksregeneration of hematopoiesis occurred much more rapidly due toproliferation of progenitors from the adherent layer. Moreover, thecells from the experimental group when plated in agar grew only in thepresence of IL-3 giving about 100 CFU per 50,000 cells; no leukemic cellgrowth was observed at least during 4 weeks. Thus, marrow treated exvivo with an effective dose of AraC in combination with INPROL can bepurged of cancerous cells while the stem cells are be protected. Itshould be possible to extend this modality to other forms ofchemotherapy or radiation treatments.

2. Marrow Repopulating Ability (MRA) and Thirty Days Radioprotection areIncreased by INPROL Treatment in Vitro.

MRA, the ability of cells to repopulate the bone marrow of lethallyirradiated mice, together with the ability to confer radioprotection for30 days, is a direct in vivo measurement of the potential to rescuemyelosuppressed animals (Visser et al. Blood Cells 14:369-384, 1988).

For radioprotection studies BDF1 mice were irradiated with 9.5Gy andrestored by transplantation of bone marrow from testosterone-stimulateddonors. One group of recipients was restored by bone marrow cellspreincubated for 4 hours with medium (controls—group A) and another(group B) with 25 ng/ml pINPROL. Cells in both groups were washed and30,000 cells per mouse were transplanted into irradiated animals. Thesurvival data are shown (FIG. 11). The sum of 3 experiments is depicted,with controls normalized to 100%. pINPROL incubation increased thesurvival of mice from 36.5% in control group up to 61.8% by Day 30.

The increase of MRA induced by preincubation with INPROL could be one ofthe mechanisms in the improving of the radioprotection. To examine thishypothesis, MRA was measured according to Visser et al. (op. cit.).Briefly, the donor BDF1 mice were pretreated with testosterone, theirbone marrow was preincubated with medium or pINPROL for 4 hours andinjected into irradiated animals. On Day 13, the bone marrow cells fromrecipient femurs were plated in agar in 3 different concentration (0.01,0.05, 0.1 equivalent of a femur) in the presence of 20% of horse serumand 10% of WEHI-CM. The number of Day 7 colonies represented the MRA asfar as the colony-forming cells in the bone marrow of recipients at thetime were the progenitors of the donor's immature stem cells.

As can be seen on FIG. 12 the MRA of the preincubated with INPROL cellpopulation is greater than in the control group (B).

EXAMPLE 9 Antihyperproliferative Effect of INPROL on Stem Cells CanChange Their Differentiation Abnormalities

Hyperproliferation of CFU-S is not only seen during restoration fromcytotoxic drugs or irradiation but also as a consequence of normalaging, and is thought to be a major feature in Myelodysplastic Syndrome(MDS). It is accompanied by the differentiation disturbances such as aprevalence of the erythroid differentiation while the differentiationalong the granulocytic pathway is reduced.

Bone marrow cells were incubated for 4 hours at 37° C. with 25 ng/ml ofpINPROL or media (Control), washed and then plated in agar with 20% ofhorse serum, 2 U/ml Erythropoietin, and 10% WEHI-CM. The number of BFU-Eand GM-CFU colonies were scored on Day 7. Data presented in Table 4 aresummarized from 3 experiments—4 animals per point were taken for eachgroup; 4 dishes were plated.

As is obvious from Table 4, the incubation of normal bone marrow (NBM)from intact young animals (BDF1 8-12 weeks old) with INPROL did notchange the number or proportion of different types of colonies. BDF₁donors pretreated with Testosterone Propionate (TSP) showed the sameincrease in CFU-S proliferation as was seen before (Example 1, 3, 4) aslight increase in the erythroid progenitor number (BFU-E colonies) anda decrease in GM-CFU, which were completely abrogated by the incubationwith INPROL. In addition, the abnormally high level of CFU-Sproliferation was returned to 10% of CFU-S in S-phase of cell cycle.CEFU-S hyperproliferation is known to be a feature of some mouse strainssusceptible to viral leukemia induction, for example Balb/c mice (Table4), and can also be observed in older animals (Table 4). The sameredistribution of committed progenitors seen in TSP treated BDF1 mice isobserved in Balb/c and in older (23-25 month old) BDF1, which have incommon the abnormally high level of CFU-S proliferation. The correctionof both the proliferation of CFU-S and the differentiation was inducedby the incubation with INPROL. What is even more clinically relevant,the study showed that the in vivo injection of INPROL (2 μg/mouse)affected both proliferation of CFU-S and the ratio of erythroid (BFU-E)and GM-colonies (Table 4).

TABLE 4 INPROL Effect On CFU-S Differentiation Into CommittedProgenitors BFU-E and CFU-GM Percent Donors Of CFU-S Bone Killed byMarrow pINPROL ³HTdR BFU-E CFU-GM BDF₁ Young − 12.0 ± 0.3 28.33 ± 1.9146.22 ± 3.44 + 15.0 ± 1.3 22.00 ± 3.74 47.70 ± 3.72 BDF₁ Old − 47.1 ±1.9 43.75 ± 1.54  24.0 ± 1.33 + 11.4 ± 0.7 15.25 ± 1.45  44.0 ± 7.63BDF₁ − 53.2 ± 1.6 32.67 ± 2.44 15.71 ± 2.28 Stimulated +  7.2 ± 0.412.00 ± 1.83 35.50 ± 1.4  by TSP Balb/C − 57.0 ± 1.9 47.60 ± 2.96 33.57± 3.45 + 23.0 ± 2.4 24.86 ± 2.53 70.60 ± 4.96

EXAMPLE 10 Immunostimulatory Activity of INPROL

It has been observed that the incubation of bone marrow cells containinga high proportion of proliferating CFU-S with INPROL not only changesthe cycling of CFU-S, but also their differentiation, switching thepredominantly erythroid differentiation in favor of granulocytic andlymphoid progenitors. This property of INPROL is of importance due tothe immunosuppression side effects of cytotoxic chemotherapy orradiotherapy, as well as the immunosuppression accompanyinghyperproliferative stem cell disorders and aging.

The example shows the direct effect of INPROL on the differentiation ofimmature precursors from the Lymphoid Long Term Culture (LLTC)established according to Wittlock & Witte (Ann. Rev. Immun. 3:213-35,1985) into pre-B progenitors, measured by the formation of colonies inmethylcellulose containing IL-7.

LLTC were established as described and fed with fresh LLTC-media (TerryFox Labs., Vancouver, Canada) twice a week. Nonadherent cells wereharvested once a week, washed free of factors and incubated for 4 hourswith 25 ng/ml pINPROL or medium alone for control. After the incubation,the cells were washed and plated at a concentration of 10⁵ cells/ml inmethylcellulose, containing 30% FCS, and 10 ng/ml of IL-7. Data from 3weeks are shown in FIG. 13. The number of large pre-B colonies varied incontrol, increasing with time, but preincubation with INPROL alwaysstimulated the growth of colonies 4 to 8 fold above the control level.This demonstrates an immunostimulatory property of INPROL which is ofuse in correcting immunodeficient states and in increasing desiredimmune responses, e.g., to vaccination.

EXAMPLE 11 INPROL Improves Repopulating Ability of Stem Cells—Long TermCulture Initiating Cells from Patient with CML

Chronic myeloid leukemia (CML) is a lethal malignant disorder of thehematopoietic stem cell. Treatment of CML in the chronic phase withsingle-agent chemotherapy, combination chemotherapy, splenectomy, orsplenic irradiation can control clinical signs and symptoms, but doesnot significantly prolong survival. As CML progresses from the chronicto the accelerated stage, standard therapy is not effective. At present,bone marrow transplantation (BMT) is the only known curative therapy forCML. Therapy with unrelated donor BMT is difficult due tohistoincompatibility problems. Fewer than 40% of otherwise eligible CMLpatients will have a suitably matched related donor, thereforeautologous transplantation is preferred. Ex vivo conditioning ofautologous BMT for infusion together with the ability to selectnon-leukeniic (Ph-negative) myeloid progenitors from Ph-positivepatients growing in Long Term Culture (LTC) suggest the potential ofautologous sources of normal stem cells to allow aggressive andeffective therapy of CML.

In the context of BMT, a hematopoietic stem cell can be defined as onehaving the ability to generate mature blood cells for extensive periods.We have used the human LTC system developed by C. Eaves & A. Eaves bothfor quantitating stem cell numbers and as a means to manipulate them fortherapeutic use. This involves seeding cells onto a pre-established,irradiated human marrow adherent layer, these cultures are thenmaintained for 5 weeks. The end point is the total clonogenic cellcontent (adherent+non-adherent) of the cultures harvested at the end ofthis time. Clonogenic cell output under these conditions is linearlyrelated to the number of progenitors (Long Term Culture Initiating Cells(LTC-IC)) initially added; the average output from individual humanLTC-IC is 4 clonogenic progenitors per LTC-IC. It has been shownpreviously that when marrow from patients with CML is placed undersimilar conditions, leukemic (Ph-positive) clonogenic cells rapidlydecline. By using quantitation of residual normal LTC-IC, in patientswith CML it is possible to select those likely to benefit from intensivetherapy supported by transplantation of cultured autografts (Phillips etal., Bone Marrow Transplantation 8:477-487, 1991).

The following procedure was used to examine the effect of INPROL on thenumber of clonogenic cells (LTC-IC) among bone marrow transplant cellsestablished from the peripheral blood of a patient with CML.

Cultures were initiated as long term cultures on pre-irradiated stroma.The. peripheral blood of a healthy donor was used as the control.Peripheral blood cells from a CML patient were preincubated with orwithout pINPROL (25 ng/ml) for 4 hours, washed and placed in the LTC-ICsystem for 5 weeks to determine the control number of LTC-IC. Forexperiments, other, parallel cultures were established for 10 days. Themixture of adherent and non-adherent cells from cultures growing for 10days was preincubated with or without pINPROL and placed onpre-established feeders for an additional 8 weeks. The number of LTC-ICfrom each experimental culture was estimated by plating both theadherent and non-adherent cells in methylcellulose with the appropriategrowth factors (Terry Fox Laboratories, Vancouver, Canada) and countingthe resulting total number of colony forming cells. The LTC-IC valuesobtained using this procedure were derived from assessment of the totalclonogenic cells (CFC) content using the formula:

#LTC-IC=#CFC/4

Data presented on FIG. 14 show that there was no loss in LTC-IC duringthe first 10 days of culture initiated from the healthy donor's bonemarrow and approximately 30% of the number of input LTC-IC were stillpresent after 5 weeks in culture. The number of the CML patient's LTC-ICwas drastically reduced to about 8% during the 10 day period and did notrecover during further incubation, while the preincubation of cells withINPROL increased the LTC-IC level to 30% of initial number and it wasmaintained during 8 weeks.

Clinically relevant applications of INPROL predicted by thesepreliminary data include their use in strategies for selectivelyimproving the normal stem cell content of fresh or cultured marrowtransplants, strategies for enhancing the recruitment of residual normalstem cells in vivo also protocols for transferring new genetic materialinto human marrow stem cells for the further transplantation intopatients.

EXAMPLE 12A A Method of Isolation of Immunoactive Inhibitor ofProliferation of Stem Cells from Bone Marrow Preparations

The bone marrow was isolated from pigs' ribs. The ribs from the pigs'carcasses were separated and cleaned from the muscle fibers and fat, cutinto pieces and the bone marrow was extracted by a hydropressmanufactured by the Biophyzpribor. The bone marrow cells are separatedby centrifugation in a centrifuge K-70 at 2,000 rpm for 20 minutes. Theextract supernatant is then successively subjected to ultrafiltrationthrough Amicon USA membranes XM-100, PM30, PM-50. According to theanalysis by electrophoresis, the main component of the product isalbumin (see FIG. 1).

Biochemical Purification

The bone marrow extract and protein components of the fractions wereanalyzed at every step of purification by gel electrophoresis in 10%polyacrylamide, containing 0.1% sodium dodecyl sulfate. Up to 7% ofsodium dodecyl sulfate and up to 0.5-1% of mercaptoethanol were added tothe samples which were incubated for 5 minutes at 70° C. prior toloading on the gel.

The electrophoresis was conducted at 20Y cm of the gel for five hours.Then the gel was stained in 0.25% Coomassie CBBC250 in a mixture ofethanol:wateracetic acid 5:5:1 for one hour at 20° C. and washed inseveral changes of 7% acetic acid. The activity of the product wasevaluated by the method of inhibition of proliferation of stemhematopoietic cells (CRU-S). The method is detailed hereafter.

Stage 1. Purification by Precipitation with Ammonium Sulfate.

The activity was purified by precipitation with ammonium sulfate at 25%with saturation of 40 to 80% which was selected based on the results inTable 5.

TABLE 5 Saturation (%) 0-40 40-60 60-80 80-100 Activity (%) 37.2-35.437.2-1.8 37.2-12.8 37.2-26.1 =1.8% =35.4% =24.4% =11.1%

The amount of the preparation used for testing after each step ofpurification was determined in accordance with the level of purificationand equivalent to the dose of 2×10^(—2) mg of the initial productActivity was determined by the formula:

% Change=%Sa-%Sb where %Sa is %S in control %Sb is %S after incubationwith the test fraction.

The fraction was desalted in order to lower the concentration ofammonium sulfate 20 times before each testing of activity and beforeeach following purification step.

Stage 2. The impure inhibitor from Stage 1 is applied after desaltingand fractionated utilizig ion exchange chromatography, here DEAE 23cellulose, and then eluted with a gradient of sodium acetate buffer (pH6.0).

The active fractions of inhibitor elute between 3-5 mM.

The volume of the column was 1 ml and speed of elution was 4 ml/hour.The detection was conducted by the chromatograph Millicrome at 230 and280 nm. Fraction 1 (see FIG. 2) which exhibited the highest activity wasisolated and eluted in 5 mMK sodium acetate buffer (see Table 6).

TABLE 6 Fractions 1 2 3 4 5 Activity 46.3-0 46.3-14.1 46.3-42.146.3-19.6 46.3- 45.1 =46.3% =32.2% =4.2% =26.7% =1.2%

The electrophoresis data indicates that the main proteincontarninant—albumin (see FIG. 3) is removed from this fraction whichleads to an additional fourfold purification.

Stage 3. The partially purified inhibitor from Stage 2 is applieddirectly to a G-75 Sephadex column.

The volume of the column is 20 ml (20×1), the elution rate is 2 ml/hour.The elution buffer is 50 mM NaCl, 10 mM Tris-HCl, pH 7.5. Detection wasconducted on a chromatograph Millichrome at 230 and 280 nm. Fraction 5which had the highest activity was isolated.

TABLE 7 Fractions 1 2 3 4 5 Activity 42.2-19.1 42.2-35.2 42.2-21.5 42.2-42.2-0 38.8 =23.1% =7.0% =20.7% =3.4% =42.2%

Stage 4. Reverse-phase chromatography (Pharmacia FPLC System) utilizingPro-REC columns is performed on an Ultrasfera matrix. Protein is elutedusing 0.1% trifluoracetic acid in an acetonitrile gradient

The homogeneity of a product with MW 16-17 kD is equal to 90% as wasshown in analyzing the acrylamide/sodium dodecyl sulfate gel (see FIG.6). The result is represented in FIG. 4. Activity is determined onfraction 5. The final yield of the product is 5%. As a result, the totalamount of protein with MW 16 kD after the purification is 650 ng/ml ofthe initial product. During the purification process the product wassubmitted to heat incubation at 70° C. for several minutes but no lossof biological activity was detected.

EXAMPLE 12B Alternative Method for Isolating Larzer Quantities of INPROLInitial Isolation

Ribs from fresh pig carcasses are cleaned of muscle fibers and fat, thencut to pieces and soaked in phosphate-buffered saline in the ratio 1:1(weight/volume). The obtained mixture is crushed by hydraulic press toseparate bone marrow from solid bone material.

The suspension of bone marrow cells is collected and filtered free ofsolid particles through four layers of the cheese-cloth. The filtrate isincubated at 56° C. for 40 minutes, then cooled in an ice-bath to 4° C.The resulting precipitate is removed by centrifugation at 10,000 g for30 minutes at 4° C. and discarded.

The clarified supernatant is added dropwise during 30 minutes to 10volumes of stirred ice-cold acetone containing 1% by volume ofconcentrated hydrochloric acid. The resulting mixture is kept at 4° C.for 16 hours for complete formation of the precipitate. Then theprecipitate is pelleted by centrifugation at 20,000 g for 30 minutes at4° C. This pellet is washed with cold acetone and dried.

HPLC Purification

The pellet is dissolved in HPLC eluent buffer A containing 5%acetonitrile (MeCN) and 0.1% triflouroacetic acid (TFA) to final proteinconcentration 8-10 mg/ml. This solution (0.5-0.6 ml) is loaded onto250×4.6 mm HPLC column packed with Polisil ODS-300 (10 mm) andequilibrated with the same buffer A.

The elution is accomplished by gradient of buffer B (90% MeCN, 0.1% TFA)in buffer A at the flow rate of 1 ml/min according to the followingprogram:

Time, min % of B  0 0  4 0  5 25 25 90

An additinal step of 100% B for 5 minutes is used to wash the columnprior to equilibration. Then the column is equilibrated again forreturning it to the initial state, and the next portion of the proteinsolution can be loaded. A typical chromatogram is shown in FIG. 5.

During the separation the column effluent is monitored at 280 nm for thedetection of protein peaks. Fractions containing the protein materialare collected, separated peaks are pooled and rotary evporated as 30° C.to dryness. The obtained residues are dissolved in ditilled water andassayed by bioactivity test and by SDS-PAGE (14% gel, reducingconditions). The peak containing the active material is eluted between70 and 80% of the buffer B and contains the main protein band of 16 kDand the traces of faster moving proteins as assayed by SDS-PAGE.

An analysis of the material obtain by collecting only the second majorHPLC peak is shown in FIG. 15 (A and C). Material containing both peaks(e.g., FIG. 5) will be referred to herein as pINPROL Preparation 1 andthose consisting of only the second peak will be referred to as pINPROLPreparation 2. 500 ug of this active, purified pINPROL Preparation 2 wasloaded onto a C4 reverse phase column (Vydac) and eluted using a lineargradient of 95% acetonitrile in 0.1% trifluoroacetic acid. The materialeluted as a single peak at 53% acetonitrile (FIG. 15A). When 250 μg ofMIP-1α (R&D Systems), however, was run under identical conditions, iteluted at 43.9% acetonitrile (FIG. 15B—note that earlier peaks prior to14% acetonitrile are artifactual and due to air bubbles in thedetector). Thus, naturally occuring INPROL is substantially morehydrophobic than MIP-1α under these conditions. TGFβ is known to eluteat lower concentrations than that observed for pINPROL under theseconditions (Miyazono et al. J. Biol. Chem. 263:6407-15, 1988).

A gel of the eluted pINPROL material is shown in FIG. 15C. Lane 1 is thecrude material, Lane 2 is molecular weight markers and Lane 3 is thepurified material. There are two major bands, one at approximately 14 kDand one at approximately 35 kD. It is believed that the 35 kD band is amultimeric form of the 14 kD band.

EXAMPLE 13A Active INPROL Preparations Contain Hemoplobin Beta Chain

pINPROL was prepared as shown in FIG. 5 (i.e., pINPROL Preparation 1(see Example 12B)). The material was run on SDS-PAGE and transferred tonitrocelluose using standard techniques. The material was subjected toN-terminal sequence analysis using an ABI 477A protein sequencer with120A Online PTH-AA analyzer using standard techniques. The followingN-terminal sequence was obtained:

VHLSAEEKEAVLGLWGKVNVDEV . . . (Seq. ID NO:23)

Computer search of the protein databases reveal that this sequence hasidentity with the N-terminal sequence of the beta chain of porcinehemoglobin (cf. FIG. 16C).

EXAMPLE 13B Active INPROL Preparations Contain Hemoglobin Alpha Chain

As shown in FIG. 15C, protein purified by collecting the second majorpeak shown in FIG. 5 (i.e., pINPROL Preparation 2) resulted in two majorbands corresponding to molecular weights of approximately 15K and 30K,as well as several minor bands. SDS-PAGE gels were transferred tonitrocellulose using standard techniques and individual bands wereexcised and subjected to N-terminal sequence analysis as in EXAMPLE 13A.The following N-terminal sequence was obtained for the 15 kD band:

VLSAADKANVKAAWGKVGGQ . . . (Seq. ID NO:24)

The 30 kD band was subjected to limited proteolytic digest and thefollowing internal sequence was obtained:

**FPHFNLSHGSDQVK . . . (Seq. ID NO:25)

The first sequence shows identity with the N-terminal sequence ofporcine hemoglobin alpha chain whereas the second sequence showsidentity with residues 43-56 of the porcine hemoglobin alpha chain (seeFIG. 16C for a sequence comparison of human, murine and porcine alphaand beta hemoglobin chains). Repeat sequencing of these bands as well asof some of the minor bands consistently yielded portions of the alphaglobin sequence. Thus the various bands observed in FIG. 15C representeither fragments or aggregates of the porcine hemoglobin alpha chain.

EXAMPLE 13C Further characterizations of Porcine INPROL

In order to further compare pINPROL to porcine hemoglobin, twicecrystallized porcine hemoglobin was obtained from Sigma Chemical Companyand subjected to reverse phase HPLC as described in Example 12B for FIG.15. As can be seen in FIG. 17, the HPLC chromatogram of intacthemoglobin is similar to that seen for the pINPROL Preparation 1.Further, in a direct comparison, the pINPROL Preparation 2 shown in FIG.17A (i.e., derived from the second peak of FIG. 5) is seen to overlapwith that of the second two peaks of porcine hemoglobin (FIG. 17B), withretention times of 52.51 and 52.25 minutes for the major peaks,respectively. It should be noted that heme co-migrates with the firstmajor peak in hemoglobin, in this case at 49.153 minutes; heme istherefore a component of pINPROL Preparation 1 but not of Preparation 2.This is confirmed by the lack of absorption of this pINPROL preparationat 575 nm, a wavelength diagnostic for the presence of heme.

The predicted molecular weights of porcine hemoglobin alpha and betachains are 15038 Daltons and 16034 Daltons, respectively. As can be seenin the SDS-PAGE chromatogram in FIG. 18, the first two peaks arecomposed of the higher molecular weight chain and the second two arecomposed of the lower molecular weight chain. Thus the first two peaksappeared to represent hemoglobin beta chain and the second two peaks torepresent hemoglobin alpha chain.

Additional separations of porcine hemoglobin were carried out using ashallow elution gradient (FIG. 21). N-terminal analyses of these peaksdemonstrated that the first peak is porcine alpha chain and the secondporcine beta chain. Bioassay results confirm that both isolatedhemoglobin chains are biologically active (e.g., Examples 14 and 15).

In order to further compare pINPROL Preparation 2 and hemoglobin betachain, 2-dimensional electrophoreses were conducted (FIG. 19). As afirst dimension, isoelectric focusing was carried out in glass tubesusing 2% pH 4-8 ampholines for 9600 volt-hours. Tropomyosin (MW 33 kD,pI 5.2) was used as an internal standard; it's position is marked on thefinal 2D gel with an arrow. The tube gel was equilibrated in buffer andsealed to the top of a stacking gel on top of a 12.5% acrylamide slabgel. SDS slab gel electrophoresis was carried out for 4 hours at 12.5mA/gel. The gels were silver stained and dried.

A comparison of the 2D electrophoretic patterns revealed only one or twominor spots that are different between HPLC purified hemoglobin betachain and the pINPROL Preparation 2. Western analyses, usinganti-porcine hemoglobin antibodies and either 1D or 2D electrophoresis,confirm the presence of beta hemoglobin in the preparation. Thus theactive pINPROL Preparation 2, prepared according to Example 12B, issubstantially porcine hemoglobin beta chain.

EXAMPLE 14 Hemoglobin Alipha Chain, Hemoglobin Beta Chain or IntactHemoglobin Exhibit Stem Cell Inhibitory Activity

To confirm that hemoglobin beta chain has INPROL activity, a suicideassay using bone marrow from testosterone-treated mice was conductedusing the methodology described in Example 1 using material purified asin Example 12B. As shown in Table 8, 15% of normal mouse bone marrowcells were killed as opposed to 36% in the testosterone-treated animals.As expected, this indicated that testosterone treatment increases thepercentage of cells in cycle (thus rendering them more susceptible tokilling—e.g. Example 1). In sharp contrast, cells fromtestosterone-treated animals incubated with either pINPROL or purifiedhemoglobin beta chain at 40 ng/ml showed a dramatic lowering of thepercentage of cells in cycle from 36% to 0% and to 7%, respectively. Thehigher dose of 200 ng was less effective for both proteins. As apositive control, the previously characterized stem cell inhibitorMIP-let reduced cycling to 13%.

A similar assay can be performed in vitro, using the cycling status ofCFU-MIX instead of CFU-S. The assay is performed as described above forthe CFU-S assay except that cytosine arabinoside (Ara C, 30micrograms/ml) is used as the cycle-specific toxic agent instead of highdose tritiated thymidine (see B. I. Lord in Haemopoiesis—A PracticalApproach, N. G. Testa and G. Molineux (Eds.), IRL Press 1993; Pragnellet al. in Culture of Hematopoietic Cells, R. I. Freshney, I. B. Pragnelland M. G. Freshney (Eds.), Wiley Liss 1994) and a mouse strain with highendogenous cycling rates (Balb/c) is used instead oftestosterone-treated BDFI mice. As shown in Table 9, highly purifiedporcine beta chain, or highly purified porcine alpha chain, are bothactive in this assay. Note that in this assay, cycling levels for cellstreated with pINPROL occasionally have negative numbers, indicating thatthere are even more colonies in the Ara C treated pool than in thenon-treated pool.

As described in Example 2, pINPROL inhibits the proliferation of themurine stem cell line FDCP-MIX in a tritiated thymidine uptake assay.FIG. 20 demonstrates that purified hemoglobin alpha or beta chains areboth active in this assay, with inhibitions seen at <2 ng/ml.

The foregoing provides evidence that the beta chain of porcinehemoglobin exhibits INPROL activity. Other data (e.g., Table 9, FIG. 20)demonstrate that isolated alpha chain, as well as intact hemoglobin, arealso active as stem cell inhibitors. Active preparations also includemixtures of alpha and beta chains (e.g., FIG. 5).

The observations that isolated alpha globin chain and/or isolated betaglobin chain are active indicate that the activities described here donot require an intact three-dimensional hemoglobin structure. Fragmentsof alpha and beta chain are also active as stem cell inhibitors andstimulators.

TABLE 8 Treatment % Kill NBM¹ 15 TPBM² 36 pINPROL 200 ng/ml 23  40 ng/ml0 Hbg³ 200 ng/ml 25  40 ng/ml 7 MIP-1α 200 ng/ml 13 ¹NBM = Normal BoneMarrow ²TPBM = Bone marrow from testosterone treated mice ³Hbg = C₄Reverse-phase purified porcine hemoglobin beta chain (derived from 2Xcrystallized pig hemoglobin)

TABLE 9 Treatment % Kill Control¹ 43 Porcine alpha chain² −4 Porcinebeta chain² −14 ¹Control - Bone marrow from Balb/c mice ²100 ng/ml(Purified as in FIG. 21)

EXAMPLE 15 Purified INPROL, Purified Porcine Alpha Hemoglobin orPurified Porcine Beta Hemoglobin are Active In Vivo

In order to test the ability of purified porcine hemoglobin chains toact in vivo, BDF₁ mice were injected with testosterone propionate asdescribed in Example 1. Twenty four hours later, mice received 500 ng ofeither pINPROL, porcine hemoglobin alpha chain (purified from peripheralred blood cells as in FIG. 21), porcine beta chain (purified fromperipheral red blood cells as in FIG. 21) or the equivalent volume ofcarrier intravenously. Forty eight hours later the bone marrow from eachmouse was harvested and the CFU-MIX assay conducted as described inExample 14. As shown in Table 10, pINPROL, pig alpha chain and pig betachain all were active in vivo, reducing the percent of CFU-MIX in cycleto basal levels.

TABLE 10 Treatment % Kill Control¹ 45 pINPROL² 5 Porcine alpha chain² 5Porcine beta chain² −5 Basal³ 4 ¹Control - Bone marrow fromtestosterone-treated BDF₁ mice ²100 ng/ml ³Basal - Bone marrow fromuntreated BDF₁ mice

EXAMPLE 16 Purified Human Hemoglobin Alpha Chain, Biotinylated HumanHemoglobin Alpha Chain, Biotinylated Human Hemoglobin Beta Chain, HumanHemoglobin Gamma Chain and Human Hemoglobin Delta Chain All Exhibit StemCell Inhibitory Activity In Vitro

Human hemoglobin was obtained either from Sigma Chemical Corporation orwas isolated by standard means from adult human peripheral blood orumbilical cord blood. Individual chains were isolated by reversed-phaseHPLC in a similar manner as that described above for porcine alpha andbeta chains (see B. Masala and L. Manca, Methods in Enzymology vol. 231pp. 21-44, 1994). Purified alpha, beta, gamma and delta chains wereobtained. For biotinylated alpha and beta chains, 1 mg of adult humanhemoglobin was treated with 37 μg of NHS LC Biotin (Pierce) and thechains separated by reverse phase chromatography as above.

At As shown in Tables 11, 12 and 13, purified human alpha, biotinylatedhuman alpha, biotinylated human beta, human gamma and human deltahemoglobin chains are all active in the CFU-MIX cycling assay.

TABLE 11 Treatment % Kill Control¹ 49 Human alpha chain² −1 Human betachain² 41 Human gamma chain² −63 ¹Control - Bone marrow from Balb/c mice²100 ng/ml

TABLE 12 Treatment % Kill Control¹ 47 Human gamma chain² 12 Human deltachain² −4 ¹Control - Bone marrow from Balb/c mice ²100 ng/ml

TABLE 13 Treatment % Kill Control¹ 68 Human alpha chain² 19 Biotinylatedalpha chain² 7 Human beta chain² 55 Biotinylated beta chain² 25¹Control - Bone marrow from Balb/c mice ²100 ng/ml

EXAMPLE 17 Purified Human Alpha Chain, Alpha-Beta Dimer or Hemoglobinare Active In Vivo

Purified human alpha chain, alpha-beta dimer or hemoglobin were testedin an in vivo assay as described in Example 15. As shown in Table 14,each of these were active at a concentration of 500 ng/mouse.

TABLE 14 Treatment % Kill Control¹ 49 Human alpha chain −22 Humanalpha-beta dimer 14 Human hemoglobin −31 ¹Control - Bone marrow fromtestosterone-treated BDF₁ mice

EXAMPLE 18 Porcine INPROL is Active on Human Mononuclear or CD34⁺ CordBlood Cells In Vitro

In order to investigate the ability of purified INPROL from porcine bonemarrow to affect cycling on human progenitors, umbilical cord bloodcells were obtained. Either the total mononuclear cell fraction obtainedafter separation on Ficoll or the CD34⁺ fraction obtained afterfractionation on anti-CD34 affinity columns (CellPro Inc.) was used.Cells were incubated for 48 hours in vitro in the presence ofinterleukin 3 (IL-3) and stem cell factor (SCF) (100 ng/ml each) inorder to ensure that the early stem cells were in cycle. After thispreincubation, cycling assays were conducted as described in Example 14for the mouse except that CFU-GEMM (instead of CFU-MIX) were counted onDay 18 after plating. As shown in Table 15, porcine INPROL inhibitedcycling of CFU-GEMM in either the bulk mononuclear cells or in the CD34⁺fraction.

TABLE 15 Treatment % Kill Mononuclear Cells Control 93 pINPROL¹ 16 CD34⁺Cells Control 41 pINPROL¹ 21 ¹100 ng/ml

EXAMPLE 19 Purified Human Alpha Hemoglobin is Active on Human CFU-GEMM

Human umbilical cord blood mononuclear cells were obtained and incubatedin IL-3 and SCF and used in a cycling assay as described in Example 18.As shown in Table 16, both porcine INPROL purified from bone marrow andhuman alpha hemoglobin, purified from peripheral blood, were active inthis assay.

TABLE 16 Treatment % Kill Control 100 pINPROL¹ −6 Human alpha chain¹ −23¹100 ng/ml

EXAMPLE 20 Peptides obtained from Human Alpha Hemoglobin and from HumanBeta Hemoglobin Sequences are Active

To identify active peptide sequences, the three dimensional structure ofmyoglobin (which is inactive in this assay) was superimposed on thenative three dimensional structure of the alpha chain present in adulthuman hemoglobin using a computer modeling program. Two peptides(representing amino acids 43-55 and 64-82, which are regions which arestructurally different from myoglobin in three-dimensional space) wereidentified as having activity in the CFU-MIX cycling assay. In order tomore closely approximate the loop found in the native alpha chain, acyclic derivative of the 43-55 peptide (c43-55) (utilizing a disulfidebond) was also synthesized and found to be active.

The sequence of these peptides is as follows:

43-55 Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (Seq. ID No:1)

(“Peptide 43-55”)

c(43-55) Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys(Seq. ID No:2)

(where the two Cys residues are disulfide-bonded)

(“Cyclic Peptide 43-55”)

64-82Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(Seq. ID No:3)

(“Peptide 64-82”)

Two hemorphin sequences, hemorphin 10 (amino acids 32-41 of the betachain sequence) and hemorphin 7 (amino acids 33-40) were tested andfound to be active. The sequences are as follows:

Hermorphin 10 Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:26)

Hemorphin 7 Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:27)

To test the activity of these sequences, the CFU-MIX cycling assay wasconducted as described in Example 14. As shown in Tables 17-19, thesepeptides all are active in this assay.

TABLE 17 Treatment % Kill Control 47 pINPROL¹ 0 Peptide (43-55) 100ng/ml 2  10 ng/ml 18  1 ng/ml 11 ¹100 ng/ml

TABLE 18 Treatment % Kill Control 43 Peptide (43-55)¹ 5 Peptide (64-82)¹9 Hemorphin 10¹ 1 Hemorphin 7¹ 0 ¹All peptides tested at 100 ng/ml

TABLE 19 Treatment % Kill Control 47 Cyclic Peptide 43-55¹ 0 ¹Tested at100 ng/ml

EXAMPLE 21 A Peptide Fragment Obtained from Human Alpha Hemoglobin byFormic Acid Cleavage is Active

Human alpha hemoglobin chain has a formic acid cleavage site betweenamino acid positions 94 and 95 (Asp-Pro). Cleavage was obtained byincubating purified human alpha chain (as in Example 16) at aconcentration of 1 mg/ml in 70% formic acid for 72 hours at 37° C. The1-94 fragment was purified from the uncleaved alpha chain and the 95-141fragment by reverse-phase HPLC as in Example 16; fractions were followedusing SDS-PAGE (as in Example 22). Identity of the purified 1-94 proteinfragment was confirmed by electrospray ionization mass spectrometry.

To assess the stem cell inhibitory activity of this fragment, theCFU-MIX cycling assay is used as in Example 14:

TABLE 20 Treatment % Kill Control¹ 50 Human Alpha² 12 1-94 fragment³ 0¹Balb/c bone marrow ²Purified, non-recombinant human alpha hemoglobin,as in Example 16 (100 ng/ml) ³Purified formic-acid cleaved protein, asin the present Example (100 ng/ml)

EXAMPLE 22 Expression of Hemoglobin Alpha Chain, Polypeptide 1-141,Polypeptide 1-97, Peptide 43-55 and Peptide c(43-55) in E. coli asUbiquitin Fusions

Genes for peptides 43-55 (“p13”) and c43-55 (“p15”) (as in Example 20)were synthesized by annealing the corresponding oligonucleotidesaccording to the optimal E. coli codon usage (Anderssen and Kurland,Micro. Reviews 54:198-210, 1990). The gene for the intact human alphahemoglobin chain (“p141”) was obtained by designing a set of oligos toPCR amplify from a human bone marrow cDNA pool (Clontech, Palo Alto,Calif.). The gene for the 1-97 fragment (“p1-97”) was obtained by PCRamplification of the plasmid containing the p141 gene after appropriatesubcloning.

The above genes were expressed as ubiquitin fusion proteins (see U.S.Pat. Nos. 5,132,213; 5,196,321 and 5,391,490 and PCT WO 91/17245). Thehost strain, E. coli DH5αF′IQ (Life Technologies, Inc., Gaithersburg,Md.) was transformed with the ubiquitin expression vector, pDSUb,containing the appropriately synthesized gene (above). pDSUb is aderivative of pDS78/RBSII that expresses human ubiquitin (FIG. 22A)(Hochuli et al., Biotechnology 6:1321-5, 1988). Loetscher et at. (JBC266:11213-11220, 1991) modified pDS78/RBSII by excising thechloramphenicol acetyl transferase (CAT) sequences from the Hochuliplasmid and religating the plasmid (FIG. 22B). A synthetic ubiquitingene was constructed by pairwise annealing of kinased syntheticoligonucleotides encoding human ubiquitin with codon usage optimized forbacterial expression. pDSUb was then constructed by inserting thesynthetic ubiquitin gene, comprised of assembled oligonucleotides, intoa Klenow blunted Bam H1-Bgl II digest of the derivatized pDS78/RBSII.The resulting plasmid, pDSUb (FIG. 22C), was shown to express ubiquitinat a high level in E. coli.

The plasmid containing p97 and the one containing p141 were constructedby inserting Afl II -Pst I digested PCR products encoding the p97 orp141 protein and fusion junction, in a directional cloning, into pDSUbthat had been digested with Afl II and Pst I. Similarly, the plasmidcontaining p13 and the one containing p15 were constructed by insertingkinased and annealed oligonucleotides, bearing the appropriate stickyends and encoding the peptide and fusion junction, into Afl II -Pst Idigested pDSUb.

Transformants were selected with 100 μg/ml ampicillin, 5 μg/ml neomycin,with colonies appearing after two days at 30° C. Transformants werescreened by PCR across the insertion site. Colonies containing thecorrectly sized insert were then screened for expression of a fusionprotein of the appropriate size by SDS-PAGE (see below). The ubiquitinfusion was overexpressed by the addition of IPTG which titrates the lacrepressor, removing it from the promoter of pDSUb (DH5αF′IQ contains anupregulated lacIq gene on the F′ factor which is selected with 10 μg/mlneomycin.)

Plasmid DNA from clones that exhibited an overexpressed, inducedubiquitin fusion protein was prepared and sequenced by the dideoxymethod using the Sequenase IVersion 2.0 kit (United States Biochemical.)Positive clones were then frozen down and stored in glycerol at −80° C.Positive clones were maintained on LB plates containing ampicillin (100μg/ml), neomycin (10 μg/ml) and 1% glucose, at 30° C. They were streakedweekly for up to 10 passages, after which a fresh streak was taken froma frozen seed vial for serial culture, to insure strain authenticity.

To obtain protein for assay, 100 ml starter cultures in 250 ml shakeflasks were grown from single colonies by overnight incubation (16-20hours) in 2×YT medium with ampicillin (100 μg/ml), neomycin (10 μg/ml)and 1% glucose. Shaker flask cultures were maintained at 30° C. and 250rpm in a New Brunswick environmental shaker incubator. The next morningthe culture was diluted to liter with medium Cells were induced by IPTGaddition to 1 mM (final dilution) at OD₆₀₀=0.5 and harvested atOD₆₀₀=0.8 by centrifugation. The harvested cells were resuspended inhypotonic lysis buffer (100 μl of 50 mM Tris, pH 10.0). The bacterialcells were lysed by subjecting the suspension to three cycles offreeze-thaw (dry ice-ethanol bath for freezing and 60° C. for thawing).The suspension was then sonicated for 10 min and centrifuged at 12,000 gfor 10 min. The resulting supernatant was designated as “S1”. The cellpellet was resuspended in 50 mM Tris, pH10 and 2×SDS tricine loadingbuffer (Novex, San Diego, Calif.) (1:1). The mixture was then heated at95° C. for 15 min and centrifuged at 12,000 g for 10 min. The portion ofthe precipitate capable of being resolubilized in this manner was called“P1”. The portion of the precipitate derived from the remaining pelletwas called “P2”. P2 was resuspended in loading buffer as for P1. Samplesfrom S1, P1 and P2 were analyzed by SDS-PAGE.

SDS-PAGE gels were run using a two buffer tricine system in a minigelapparatus, with 10-20% tricine gels (Novex). Anode (bottom) buffer was0.2 M Tris, pH 9.0. Cathode (top) buffer was 0.1 M Tris, 0.1 M Tricine,0.1% SDS, pH 8.25. A commercial molecular weight marker, “Multi-Mark”(Novex) was used. Bovine ubiquitin, used as a standard, was purchasedfrom Sigma. Gels were run at a constant current of 4 mA until the dyemarker reached the bottom of the gel. Gels were stained with 0.25%Coomassie Blue R250 (Sigma) in acetic acid:methanol (10%:40%) anddestained in the same solution minus the dye.

The majority (>70%) of the intact p141-ubiquitin fusion protein wasfound in the precipitate (P1 and P₂) after centrifugation of thebacterial lysate. In sharp contrast, the majority (>70%) of thep97-ubiquitin fusion protein was found in the soluble fraction (S₁).This confirmed that the removal of the C-terminal hydrophobic regionresulted in a product with improved solubility characteristics.Similarly, the p13 and p15 peptides were also contained in the solublefraction.

The UCH-L3 ubiquinase enzyme (Recksteiner, M. (Ed.) Ubiquitin, PlenumPress (NY) 1988; Wilkinson et al., Science 246:670-73, 1989) wasexpressed in pRSET (Invitrogen, San Diego, Calif.) which was used totransform the host strain BL21/DE3. UCH-L3 is a ubiquitin-specificprotease that cleaves at the ubiquitin C-terminal extension. It waspartially purified from bacterial lysates by a 35% (w/v) ammoniumsulfate precipitation. The exact percentage of ammonium sulfate used wasmonitored by SDS-PAGE for the presence of a 25.5 kD band. Thesupernatant was dialyzed against 50 mM Tris, pH 7.4, and assayed againsta ubiquitin peptide fusion substrate. The active supernatant wasaliquoted and frozen at −20° C. A typical reaction mixture contains 3 μllysate, 1 μl 1M DTT, 1 μl UCH-L3 (as above) and 5 μl reaction buffer (50mM Tris, pH 7.4). The reaction was carried out at room temperature for20 min. For large scale digestion, 300 μl lysate was mixed with 100 μl1M DTT, 20 μl UCH-L3 and 580 μl reaction buffer.

Peptides or proteins contained in the soluble (S₁) fraction were furtherpurified by reverse phase HPLC as in Example 16; fractions weremonitored by SDS-PAGE and their identity confirmed by electrosprayionization mass spectrometry (see below). The purified peptides orproteins were enzymatically digested by UCH-L3 as above, resulting in anon-ubiquinated final product. This cleaved material was thenre-purified by reverse phase HPLC. Purification was followed by SDS-PAGEand the identity of the final product confirmed by electroprayionization mass spectrometry.

An alternative to the in vitro cleavage with UCH-L3 as described aboveis to co-express a ubiquitin cleaving enzyme in the same bacteria as thedesired ubiquitin fusion. For this purpose, a vector (pJT184) expressingthe ubiquinase UBP1 (Tobias and Varshavsky, JBC 266:12021-12028, 1991)was used. Bacteria co-expressing p97 ubiquitin fusion and UBP1 exhibitedcomplete digestion of the fusion protein in vivo; bacteria co-expressingp141 ubiquitin fusion and UBP1 exhibited partial (approximately 70%)digestion of the fusion protein. The in vivo digested p97 protein waspurified by ammonium sulfate precipitation followed by reverse-phaseHPLC as above.

To confirm the identity,of the expressed and purified polypeptides,electrospray ionization mass spectrometry was performed using a VGBiotech BIOQ instrument with quadrupole analyser. Myoglobin was used tocalibrate the instrument. The major component obtained with purified p97was a single peak of molecular weight of 10,339 daltons; this comparesfavorably with the calculated molecular weight of 10,347, confirming theidentity of the recombinant p97 fragment

EXAMPLE 23 Recombinant p1-97 Retains Stem Cell Inhibitory Activity

To assess the bioactivity of recombinant p1-97, the CFU-GEMM cyclingassay was used as in Example 18:

TABLE 21 Treatment % Kill Control¹ 62 Human Alpha² 11 p97³ 0 ¹Human bonemarrow mononuclear cells ²Purified, non-recombinant human alphahemoglobin, as in Example 16 (100 ng/ml) ³Purified recombinant p97, asin Example 22 (100 ng/ml)

EXAMPLE 24 Human Alpha Hemoglobin and Peptide 43-55 Inhibit CFU-MIXCycling In Vivo in Testosterone or 5-Fluorouracil (5FU) Treated Mice

To assess the stem cell inhibitory activity of peptide 43-55 in vivo,B6D2 F₁ mice were pre-treated with testosterone propionate as describedin Example 1 or with 5FU. Specifically, testosterone propionate (100mg/kg body weight) was injected i.p. on Day 0 into mice. Alternatively,mice were injected on Day 0 with 5FU (200 μg/kg body weight).

24 hours later (Day 1), varying doses of peptide 43-55 or vehicle wereinjected i.v. Bone marrow was harvested on Day 2 and the CFU-MDC cyclingassay conducted as in Example 14. Specifically, mice were sacrificed andsingle cell bone marrow suspensions prepared from the femurs. The cellswere washed once and the concentration adjusted to 5×10⁶ cells/ml inFischer medium. For each test condition, one milliliter of cells wasadded to each of two polypropylene tubes. The tubes were incubated at37° C. for 3 hours without (“Control”) or with (“Experimental”) theappropriate concentration of test substance. At the end of theincubation, 30 μg/ml of cytosine arabinoside (“Ara C” (Sigma)) was addedto half of the tubes and the same volume of Fischer's medium was addedto the others. The tubes were incubated for a further 1 hour at 37° C.,after which they were placed on ice and washed twice with cold Fischermedium.

The cells were readjusted to 5×10⁴-10⁶/ml in Fischer's medium and 0.5 mlof cell suspension added to 5 ml of Methocult M3430 methylcellulosemedium (Stem Cell Technologies, Vancouver, British Columbia). Themixture was vigorously mixed with a vortex and 1 ml was dispensed intoeach of five 35 mm dishes. The 35 mm dishes were in turn placed in acovered 150 mm dish with one open 35 mm dish containing sterile water.CFU-MIX colonies were counted with the use of an inverted microscopeafter 7 days of incubation at 37° C.

Differences in colony number between the tubes treated with mediumversus the tubes treated with Ara C represent the percentage of cells incycle under that condition according to the formula:

${\% \quad S} = {\frac{a - b}{a} \times 100\%}$

where

a=Number of CFU-MIX from tube incubated with medium alone

b=Number of CFU-MIX from tube incubated with Ara C

TABLE 22¹ Treatment % Kill Control 35 Human Alpha Chain (500 ng)² 13Peptide 43-55 (0.5 ng)² 0 ¹Testosterone-pretreated animals ²Amountinjected i.v. per 20 gram mouse

TABLE 23¹ Treatment % Kill Control 62 Human Alpha Chain (500 ng)² 0Peptide 43-55 (0.5 ng)² 3 Cyclic Peptide 43-55 (0.5 ng)² 14¹5FU-pretreated animals ²Amount injected i.v. per 20 gram mouse

EXAMPLE 25 Peptide 43-55 is Active as a Stem Cell Inhibitor whenBiotinylated at the N-terminal Phe (Phe₄₃) or Iodinated at Phe₄₃ orPhe₄₆

Peptide 43-55 was synthesized by solid phase peptide synthetictechniques (American Peptide Co., Sunnyvale, Calif.). Peptide analogswere synthesized with iodine at the para position of Phe₄₃ or of Phe₄₆.Biotinylated Peptide 43-55 was synthesized by linking the COOH of biotinwith a C₄ carbon linker to the N-terminal NH₂ of Phe₄₃.

TABLE 24 Treatment % Kill Control 31 Peptide 43-55 (1 ng/ml) 8Biotinylated peptide 43-55 (1 ng/ml) 15

EXAMPLE 26 Morphine Inhibits Cycling of Murine CFU-MIX In Vitro

Morphine was tested in the CFU-MIX cycling assay using bone marrow fromBalb/c mice as in Example 24:

TABLE 25 Treatment % Kill Control 44 Human Alpha Chain (100 ng/ml) 0Morphine (10⁻⁷M) 10 (10⁻⁹M) 15 (10⁻¹¹M) 32

EXAMPLE 27 The Opiate Peptides DAMGO and DALDA Inhibit Cycling of MurineCFU-MIX In Vitro

DAMGO and DALDA were tested in the CFU-MIX cycling assay using bonemarrow from Balb/c mice as in Example 24:

TABLE 26 Treatment % Kill Control 33 DAMGO (10⁻⁵M) 15 (10⁻⁷M) 0 (10⁻⁹M)38 DALDA (10⁻⁵M) 47 (10⁻⁷M) 0 (10⁻⁹M) 34

EXAMPLE 28 Nociceptin Inhibits Cycling of Murine CFU-MIX In Vitro

Nociceptin was tested in the CFU-MIX cycling assay using bone marrowfrom Balb/c mice as in Example 24:

TABLE 27 Treatment % Kill Control 31 Peptide 43-55 (1 ng/ml) 8Nociceptin (10⁻⁷M) 6 (10⁻⁹M) 0

EXAMPLE 29 Naloxone Antagonizes the Inhibitory Activity of Human Alphaand Delta Hemoglobin Chains, Hemorphin 10 and Peptide 43-55

The CFU-MIX cycling assay was conducted as in Example 24. Testsubstances were assayed by themselves or in the presence of naloxone(10⁻⁵-10⁻⁷M). Naloxone itself had no effect on the assay at theseconcentrations.

TABLE 28 Treatment % Kill Control 38 Naloxone¹ 36 Human Alpha (100ng/ml) 0 Human Alpha + Naloxone¹ 52 Peptide 43-55 (10 ng/ml) 6 Peptide43-55 + Naloxone¹ 50 Hemorphin 10 (100 ng/ml) 0 Hemorphin 10 + Naloxone¹36 ¹Used at 10⁻⁵M final concentration

TABLE 29 Treatment % Kill Control 32 Human Delta (100 ng/ml) 10 HumanDelta + Naloxone¹ 31 ¹Used at 10⁻⁷M final concentration

EXAMPLE 30 Low Concentrations of Naloxone Inhibit Cycling of MurineCFU-MIX

The CFU-MIX assay was conducted as in Example 24.

TABLE 30 Treatment % Kill Control 38 Human Alpha (100 ng/ml) 0 Naloxone(10⁻¹⁰M) 0

EXAMPLE 31 The mu Opiate Receptor Antagonist CTOP Antagonizes theInhibitory Activity of Human Hemoglobin Alpha Chain, Peptide 43-55 andPeptide 64-82

CTOP (H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂, with a disuwfidebetween Cys₂ and Pen₇) is an analog of somatostatin which is a mu opiatereceptor specific antagonist. Test substances were assayed by themselvesor in the presence of CTOP (10⁻⁷ M). CTOP itself had no effect on theassay at this concentration but antagonized the cell cycle inhibitioncaused by alpha hemoglobin or peptide 43-55.

TABLE 31 Treatment % Kill Control 42 CTOP¹ 36 Human Alpha (100 ng/ml) 0Human Alpha + CTOP¹ 20 Peptide 43-55 (10 ng/ml) 8 Peptide 43-55 + CTOP¹21 ¹Used at 10⁻⁷M, final concentration

EXAMPLE 32 Pretreatment with Human Alpha Chain Increases the Number ofLate-Forming Cobblestone-Forming Cells

The cobblestone assay was conducted as described by Ploemacher andcolleagues (Ploemacher et al., Blood 74:2755-63, 1989; van der Sluijs etal., Exp. Hematol. 18:893-6, 1990; Ploemacher et al., Blood 78:2527-33,1991; Ploemacher et al., J. Tiss. Cult. Meth. 13:63-68, 1991; Down andPloemacher, Exp. Hematol. 21:213-21, 1993). The cobblestone assaymeasures the appearance of groups of cells (or “cobblestones”) within amonolayer of stromal cells. Very primitive stem cells will not formcolonies in soft agar but do form cobblestones in the presence of astromal monolayer. The cells which form cobblestones are referred to as“cobblestone area forming cells” (CAFC). The more differentiated (e.g.,GM-CFC) progenitors form transient cobblestones which appear and thendisappear within the first few weeks of culture whereas more primitivestem cells (e.g., long-term repopulating cells) form cobblestones whichappear only after 4-5 weeks of culture. Thus, CAFC forming on days 7-14of culture are enriched in CFU-GM, CAFC forming on days 28-35 areenriched in CFU-MIX, and CAFC forming on days 28-35 are enriched inlong-term repopulating cells.

B6D2F1 mice were treated with testosterone propionate as in Example 24.The next day bone marrow was removed and incubated for 4 hours with orwithout human alpha hemoglobin chain (100 ng/10⁶ cells) after which theywere plated in a cobblestone assay. The assay used consists of limitingdilution long term bone marrow cultures (LTBMC) in 96-well plates. Thecultures were prepared by growing the FBMD-1 murine stromal cell line(Breems et al., Leukemia 11:142-50, 1997) until confluent; 96-wellplates plates with confluent monolayers were stored at 33° C. untilassay. Murine bone marrow cells were prepared as a single cellsuspension and the following dilutions of cells were plated per well in0.2 ml of LTBMC medium (Stem Cell Technologies, Vancouver): 27,000;9000; 3000; 1000; 333. Twenty wells were plated for each dilution percondition and distributed over two plates.

The frequency of cobblestone area forming cells (CAFC) was calculated aspreviously described (Ploemacher et al., Blood 78:2527-33, 1991;Ploemacher et al., J. Tiss. Cult. Meth. 13:63-68, 1991; Breems et al.,Leukemia 8:1095-104, 1994). The results are shown in FIG. 23.Preincubation of cycling stem cells with human alpha hemoglobin chainincreased the proportion of late-forming CAFC by approximately 5-fold.Treatment of non-cycling stem cells with alpha chain had no effect.

EXAMPLE 33 Human Alpha Hemoglobin and Peptides 43-55, Cyclic 43-55 and64-82 Inhibit Cycling of Human Cord Blood CFU-GEMM

The human cord blood CFU-GEMM cycling assay was conducted as in Example19. Specifically, mononuclear cells were isolated from human umbilicalcord blood cells and adjusted to 24×10⁴ cells/ml in IMDM tissue culturemedium supplemented with 10% FBS, 100 ng/ml kit ligand and 100 ng/mlhuman IL-3. The cells were incubated for 48 hours at 37° C.

After incubation, cells were washed and resuspended in serun-free NDM ata concentration of 10⁶ cells/ml. One ml of cells was added to each oftwo polypropylene tubes per condition and the cycling assay conducted asin Example 24 for mouse bone marrow. After the Ara C incubation cellswere washed with cold NDM and adjusted to 10,000 to 20,000 cells per 0.5ml IMDM and mixed with 5 ml Methocult H4433 (Stem Cell Technologies).Alternatively, Methocult H4435 methylcellulose medium (Stem CellTechnologies) was used in which case the cell concentration was adjustedto 2500-5000 cells per 0.5 ml IMDM. The cells were plated as in Example24 and CFU-GEMM colonies scored on days 14-18.

TABLE 32 Treatment % Kill Control 52 Human Alpha Chain (100 ng/ml) 0Peptide 43-55 (1 ng/ml) 20 (10 ng) 12 (100 ng) 5 Cyclic Peptide 43-55 (1ng/ml) 32 (10 ng) 0 (100 ng) 11 Peptide 64-82 (1 ng/ml) 21 (10 ng) 20(100 ng) 39

EXAMPLE 34 Human Alpha Hemoglobin and Peptide 43-55 Inhibits Cycling ofAdult Human Bone Marrow CFU-GEMM

CD34⁺ stem cells were obtained from Poietic Technologies (Gaithersburg,MD) after purification from human bone marrow by means of a CelUProcolumn. Cells were incubated for 48 hours with kit ligand and IL-3 andused in a CFU-GEMM cycling assay in Example 26.

TABLE 33 Treatment % Kill Control 47 Human Alpha Chain (ng/ml) 0 Peptide43-55 (ng/ml) 0

EXAMPLE 35 CFU-GEMM in Mobilized Human Peripheral Blood Actively Cycleand are Inhibitable by Human Alpha Hemoglobin, Peptide 43-55, DAMGO orMorphine

Peripheral blood was obtained from breast cancer patients undergoingperipheral stem cell mobilization with cyclophosphamide and G-CSFaccording to standard protocols. Red blood cells were removed withFicolU Hypaque (cells were diluted 1:1 with IMDM and 20 ml layered ontop of 16 ml Ficoll and centrifuged at 800 g for 30 minutes; mononuclearcells were removed from the interphase and washed twice in IMDM). In onecase mononuclear cells were stored frozen in liquid nitrogen beforeassay. The mononuclear cells were plated for the CFU-GEMM cycling assayas in Example 26 except that 2.5-5×10⁵ cells were plated per dish.

TABLE 34 Treatment % Kill Control (Patient #1) 48 Human Alpha Chain (100ng/ml) 0

TABLE 35 Treatment % Kill Control (Patient #2)¹ 67 Human Alpha Chain(ng/ml) 2 Peptide 43-55 (10 ng/ml) 0 Morphine (10⁻⁷M) 24 Morphine(10⁻⁹M) 0 DAMGO (10⁻⁷M) 15 DAMGO (10⁻⁹M) 0

TABLE 36 Treatment % Kill Control (Patient #3)¹ 29 Peptide 43-55 (0.1ng/ml) 23 Peptide 43-55 (1.0 ng/ml) 0 Peptide 43-55 (10 ng/ml) 15 ¹Fromcells stored frozen before assay

EXAMPLE 36 High Doses of Human Alpha or Beta Hemoglobin, Myoglobin,Peptide 1-97, Peptide 43-55, Peptide 64-82 Nociceptin or DALDA StimulateCycling of Ouiescent Murine Stem Cells

Microgram per milliliter doses of hemoglobin chains, myoglobin andpeptides were assayed for stimulation of quiescent stem cells. Bonemarrow was obtained from untreated B6D2F₁ tested in the CFU-MIX cyclingassay as in Example 24. Stem cells isolated from untreated B6D2F₁ miceare normally slowly cycling unless stimulated (e.g. by testosteronepropionate (cf. Example 1) or chemotherapy such as 5FU (cf. Example 4))to enter into cycle.

TABLE 37 Treatment % Kill Control 3 Human α Chain (1 μg/ml) 0 (10 μg/ml)24 (100 μg/ml) 40

TABLE 38 Treatment % Kill Control 9 Human β Chain (μg/ml) 55 HumanMyoglobin (μg/ml) 30

TABLE 39 Treatment % Kill Control 3 Human α Chain (100 μg/ml) 41 DALDA(10⁻⁵M) 24 (10⁻³M) 41 DADLE (10⁻⁵M) 0 (10⁻³M) 0

TABLE 40 Treatment % Kill Control 0 Human α Chain (100 μg/ml) 30 Peptide43-55 (10 μg/ml) 26

TABLE 41 Treatment % Kill Control 16 Peptide 1-97 (10 μg/ml) 62 Peptide1-97 (10 μg/ml) 41

TABLE 42 Treatment % Kill Control 4 Peptide 64-82 (1 μg/ml) 25Nociceptin (10⁻⁵M) 36

EXAMPLE 37 Intravenous Administration of High Dose Human AlphaHemoglobin Stimulates Cycling of Ouiescent Murine Stem Cells

Human alpha hemoglobin was injected i.v. into untreated B6D2F₁ mice. 24hours later, the mice were sacrificed, bone marrow collected from femursand the CFU-MIX assay conducted as in Example 24.

TABLE 43 Treatment % Kill Control (Untreated) 0 Medium (InjectionControl) 0 Human α Chain (150 μg/mouse) 48

EXAMPLE 38 Naloxone Antagonizes the Stem Cell Stimulatory Activity ofHigh Dose Human Alpha Hemoglobin, Peptide 43-55

TABLE 44 Treatment % Kill Control 6 Human Alpha Chain (100 ng/ml) 48Human Alpha Chain + Naloxone¹ 0 ¹Used at 10⁻⁷M final concentration

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

36 13 amino acids amino acid <Unknown> linear peptide 1 Phe Pro His PheAsp Leu Ser His Gly Ser Ala Gln Val 1 5 10 15 amino acids amino acid<Unknown> linear peptide 2 Cys Phe Pro His Phe Asp Leu Ser His Gly SerAla Gln Val Cys 1 5 10 15 19 amino acids amino acid <Unknown> linearpeptide 3 Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro AsnAla 1 5 10 15 Leu Ser Ala 10 amino acids amino acid <Unknown> linearpeptide 4 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe 1 5 10 9 amino acidsamino acid <Unknown> linear peptide 5 Leu Val Val Tyr Pro Trp Thr GlnArg 1 5 8 amino acids amino acid <Unknown> linear peptide 6 Leu Val ValTyr Pro Trp Thr Gln 1 5 7 amino acids amino acid <Unknown> linearpeptide 7 Leu Val Val Tyr Pro Trp Thr 1 5 6 amino acids amino acid<Unknown> linear peptide 8 Leu Val Val Tyr Pro Trp 1 5 5 amino acidsamino acid <Unknown> linear peptide 9 Leu Val Val Tyr Pro 1 5 7 aminoacids amino acid <Unknown> linear peptide 10 Val Val Tyr Pro Trp Thr Gln1 5 7 amino acids amino acid <Unknown> linear peptide 11 Tyr Pro Trp ThrGln Arg Phe 1 5 6 amino acids amino acid <Unknown> linear peptide 12 TyrPro Trp Thr Gln Arg 1 5 5 amino acids amino acid <Unknown> linearpeptide 13 Tyr Pro Trp Thr Gln 1 5 6 amino acids amino acid <Unknown>linear peptide 14 Arg Met Trp Met Phe Arg 1 5 423 base pairs nucleicacid single linear DNA (genomic) 15 GTGCTGTCTC CTGCCGACAA GACCAACGTCAAGGCCGCCT GGGGTAAGGT CGGCGCGCAC 60 GCTGGCGAGT ATGGTGCGGA GGCCCTGGAGAGGATGTTCC TGTCCTTCCC CACCACCAAG 120 ACCTACTTCC CGCACTTCGA CCTGAGCCACGGCTCTGCCC AGGTTAAGGG CCACGGCAAG 180 AAGGTGGCCG ACGCGCTGAC CAACGCCGTGGCGCACGTGG ACGACATGCC CAACGCGCTG 240 TCCGCCCTGA GCGACCTGCA CGCGCACAAGCTTCGGGTGG ACCCGGTCAA CTTCAAGCTC 300 CTAAGCCACT GCCTGCTGGT GACCCTGGCCGCCCACCTCC CCGCCGAGTT CACCCCTGCG 360 GTGCACGCCT CCCTGGACAA GTTCCTGGCTTCTGTGAGCA CCGTGCTGAC CTCCAAATAC 420 CGT 423 141 amino acids amino acid<Unknown> linear peptide 16 Val Leu Ser Pro Ala Asp Lys Thr Asn Val LysAla Ala Trp Gly Lys 1 5 10 15 Val Gly Ala His Ala Gly Glu Tyr Gly AlaGlu Ala Leu Glu Arg Met 20 25 30 Phe Leu Ser Phe Pro Thr Thr Lys Thr TyrPhe Pro His Phe Asp Leu 35 40 45 Ser His Gly Ser Ala Gln Val Lys Gly HisGly Lys Lys Val Ala Asp 50 55 60 Ala Leu Thr Asn Ala Val Ala His Val AspAsp Met Pro Asn Ala Leu 65 70 75 80 Ser Ala Leu Ser Asp Leu His Ala HisLys Leu Arg Val Asp Pro Val 85 90 95 Asn Phe Lys Leu Leu Ser His Cys LeuLeu Val Thr Leu Ala Ala His 100 105 110 Leu Pro Ala Glu Phe Thr Pro AlaVal His Ala Ser Leu Asp Lys Phe 115 120 125 Leu Ala Ser Val Ser Thr ValLeu Thr Ser Lys Tyr Arg 130 135 140 438 base pairs nucleic acid singlelinear DNA (genomic) 17 GTGCACCTGA CTCCTGAGGA GAAGTCTGCC GTTACTGCCCTGTGGGGCAA GGTGAACGTG 60 GATGAAGTTG GTGGTGAGGC CCTGGGCAGG CTGCTGGTGGTCTACCTTTG GACCCAGAGG 120 TTCTTTGAGT CCTTTGGGGA TCTGTCCACT CCTGATGCTGTTATGGGCAA CCCTAAGGTG 180 AAGGCTCATG GCAAGAAAGT GCTCGGTGCC TTTAGTGATGGCCTGGCTCA CCTGGACAAC 240 CTCAAGGGCA CCTTTGCCAC ACTGAGTGAG CTGCACTGTGACAAGCTGCA CGTGGATCCT 300 GAGAACTTCA GGCTGCTGGG CAACGTGCTG GTCTGTGTGCTGGCCCATCA CTTTGGCAAA 360 GAATTCACCC CACCAGTGCA GGCTGCCTAT CAGAAAGTGGTGGCTGGTGT GGCTAATGCC 420 CTGGCCCACA AGTATCAC 438 146 amino acids aminoacid <Unknown> linear peptide 18 Val His Leu Thr Pro Glu Glu Lys Ser AlaVal Thr Ala Leu Trp Gly 1 5 10 15 Lys Val Asn Val Asp Glu Val Gly GlyGlu Ala Leu Gly Arg Leu Leu 20 25 30 Val Val Tyr Pro Trp Thr Gln Arg PhePhe Glu Ser Phe Gly Asp Leu 35 40 45 Ser Thr Pro Asp Ala Val Met Gly AsnPro Lys Val Lys Ala His Gly 50 55 60 Lys Lys Val Leu Gly Ala Phe Ser AspGly Leu Ala His Leu Asp Asn 65 70 75 80 Leu Lys Gly Thr Phe Ala Thr LeuSer Glu Leu His Cys Asp Lys Leu 85 90 95 His Val Asp Pro Glu Asn Phe ArgLeu Leu Gly Asn Val Leu Val Cys 100 105 110 Val Leu Ala His His Phe GlyLys Glu Phe Thr Pro Pro Val Gln Ala 115 120 125 Ala Tyr Gln Lys Val ValAla Gly Val Ala Asn Ala Leu Ala His Lys 130 135 140 Tyr His 145 141amino acids amino acid <Unknown> linear peptide 19 Val Leu Ser Gly GluAsp Lys Ser Asn Ile Lys Ala Ala Trp Gly Lys 1 5 10 15 Ile Gly Gly HisGly Ala Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met 20 25 30 Phe Ala Ser PhePro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Val 35 40 45 Ser His Gly SerAla Gln Val Lys Gly His Gly Lys Lys Val Ala Asp 50 55 60 Ala Leu Ala SerAla Ala Gly His Leu Asp Asp Leu Pro Gly Ala Leu 65 70 75 80 Ser Ala LeuSer Asp Leu His Ala His Lys Leu Arg Val Asp Pro Val 85 90 95 Asn Phe LysLeu Leu Ser His Cys Leu Leu Val Thr Leu Ala Ser His 100 105 110 His ProAla Asp Phe Thr Pro Ala Val His Ala Ser Leu Asp Lys Phe 115 120 125 LeuAla Ser Val Ser Thr Val Leu Thr Ser Lys Tyr Arg 130 135 140 146 aminoacids amino acid <Unknown> linear peptide 20 Val His Leu Thr Asp Ala GluLys Ala Ala Val Ser Cys Leu Trp Gly 1 5 10 15 Lys Val Asn Ser Asp GluVal Gly Gly Glu Ala Leu Gly Arg Leu Leu 20 25 30 Val Val Tyr Pro Trp ThrGln Arg Tyr Phe Asp Ser Phe Gly Asp Leu 35 40 45 Ser Ser Ala Ser Ala IleMet Gly Asn Ala Lys Val Lys Ala His Gly 50 55 60 Lys Lys Val Ile Thr AlaPhe Asn Asp Gly Leu Asn His Leu Asp Ser 65 70 75 80 Leu Lys Gly Thr PheAla Ser Leu Ser Glu Leu His Cys Asp Lys Leu 85 90 95 His Val Asp Pro GluAsn Phe Arg Leu Leu Gly Asn Met Ile Val Ile 100 105 110 Val Leu Gly HisHis Leu Gly Lys Asp Phe Thr Pro Ala Ala Gln Ala 115 120 125 Ala Phe GlnLys Val Val Ala Gly Val Ala Thr Ala Leu Ala His Lys 130 135 140 Tyr His145 141 amino acids amino acid <Unknown> linear peptide 21 Val Leu SerAla Ala Asp Lys Ala Asn Val Lys Ala Ala Trp Gly Lys 1 5 10 15 Val GlyGly Gln Ala Gly Ala His Gly Ala Glu Ala Leu Glu Arg Met 20 25 30 Phe LeuGly Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asn Leu 35 40 45 Ser HisGly Ser Asp Gln Val Lys Ala His Gly Gln Lys Val Ala Asp 50 55 60 Ala LeuThr Lys Ala Val Gly His Leu Asp Asp Leu Pro Gly Ala Leu 65 70 75 80 SerAla Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro Val 85 90 95 AsnPhe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala Ala His 100 105 110His Pro Asp Asp Phe Asn Pro Ser Val His Ala Ser Leu Asp Lys Phe 115 120125 Leu Ala Asn Val Ser Thr Val Leu Thr Ser Lys Tyr Arg 130 135 140 146amino acids amino acid <Unknown> linear peptide 22 Val His Leu Ser AlaGlu Glu Lys Glu Ala Val Leu Gly Leu Trp Gly 1 5 10 15 Lys Val Asn ValAsp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu Leu 20 25 30 Val Val Tyr ProTrp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu 35 40 45 Ser Asn Ala AspAla Val Met Gly Asn Pro Lys Val Lys Ala His Gly 50 55 60 Lys Lys Val LeuGln Ser Phe Ser Asp Gly Leu Lys His Leu Asp Asn 65 70 75 80 Leu Lys GlyThr Phe Ala Lys Leu Ser Glu Leu His Cys Asp Gln Leu 85 90 95 His Val AspPro Glu Asn Phe Arg Leu Leu Gly Asn Val Ile Val Val 100 105 110 Val LeuAla Arg Arg Leu Gly His Asp Phe Asn Pro Asp Val Gln Ala 115 120 125 AlaPhe Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His Lys 130 135 140Tyr His 145 23 amino acids amino acid <Unknown> linear peptide 23 ValHis Leu Ser Ala Glu Glu Lys Glu Ala Val Leu Gly Leu Trp Gly 1 5 10 15Lys Val Asn Val Asp Glu Val 20 20 amino acids amino acid <Unknown>linear peptide 24 Val Leu Ser Ala Ala Asp Lys Ala Asn Val Lys Ala AlaTrp Gly Lys 1 5 10 15 Val Gly Gly Gln 20 14 amino acids amino acid<Unknown> linear peptide 25 Phe Pro His Phe Asn Leu Ser His Gly Ser AspGln Val Lys 1 5 10 8 amino acids amino acid <Unknown> linear peptide 26Val Val Tyr Pro Trp Thr Gln Arg 1 5 4 amino acids amino acid <Unknown>linear peptide 27 Tyr Pro Trp Thr 1 4 amino acids amino acid <Unknown>linear peptide 28 Gly Tyr Pro Tyr 1 4 amino acids amino acid <Unknown>linear peptide 29 Gly Lys Pro Tyr 1 4 amino acids amino acid <Unknown>linear peptide 30 Gly Leu Pro Tyr 1 6 amino acids amino acid <Unknown>linear peptide 31 Lys Met Trp Met Phe Arg 1 5 8 amino acids amino acid<Unknown> linear peptide 32 Val Arg Arg Met Phe Gly Gly Tyr 1 5 6 aminoacids amino acid single linear peptide 33 Phe Leu Gly Phe Pro Thr 1 5 6amino acids amino acid single linear peptide Modified-site /product=“position 1” /label= Xaa /note= “pyroGln” 34 Xaa Gln Gln Asp Cys Lys 1 54 amino acids amino acid single linear peptide Modified-site /product=“position 1” /label= Xaa /note= “N-AcetylSer” 35 Xaa Asp Lys Pro 1 13amino acids amino acid single linear peptide Modified-site /product=“position 1” /label= Xaa /note= “biotin-Phe, Phe or iodo-Phe”Modified-site /product= “position 4” /label= Xaa /note= “Phe, iodo-Phe”36 Xaa Pro His Xaa Asp Leu Ser His Gly Ser Ala Gln Val 1 5 10

What is claimed is:
 1. A method of stimulating stem cell proliferationcomprising contacting hematopoietic cells with a stem cell proliferationstimulating amount of INPROL or an opiate compound or a stem cellproliteration stimulating amount of a combination of INPROL and anopiate compound, wherein said INPROL is selected from the groupconsisting of a polypeptide having the sequence of amino acids 1-97 ofthe human alpha hemoglobin chain, a polypeptide having the sequence ofamino acids 1-94 of the human alpha hemoglobin chain,Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1),Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) (where the two Cys residues form a disulfide bond),Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(SEQ ID NO:3), Phe-Leu-Gly-Phe-Pro-Thr (SEQ ID NO:33),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:4),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:5),Leu-Val-Val-Tyr-Pro-Trp-Thr (SEQ ID NO:6), Leu-Val-Val-Tyr-Pro-Trp-Thr(SEQ ID NO:7), Leu-Val-Val-Tyr-Pro-Trp (SEQ ID NO:8).Leu-Val-Val-Tyr-Pro (SEQ ID NO:9), Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ IDNO:10), Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:11),Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:12), Tyr-Pro-Trp-Thr-Gln (SEQ IDNO:13), and Tyr-Pro-Trp-Thr (SEQ ID NO:27): wherein said stem cells arecells which can generate multiple lineages or other stem cells.
 2. Amethod as in claim 1 wherein said INPROL is selected from the groupconsisting of a polypeptide having the sequence of amino acids 1-97 ofthe human alpha hemoglobin chain, and a polypeptide having the sequenceof amino acids 1-94 of the human alpha hemoglobin chain.
 3. A method asin claim 1 wherein said INPROL is selected from the group consisting ofpeptides having the sequence:Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1),Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) (where the two Cys residues form a disulfide bond),Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(SEQ ID NO:3), Phe-Leu-Gly-Phe-Pro-Thr (SEQ ID NO: 33),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:4),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:5),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:6),Leu-Val-Val-Tyr-Pro-Trp-Thr (SEQ ID NO:7), Leu-Val-Val-Tyr-Pro-Trp (SEQID NO:8), Leu-Val-Val-Tyr-Pro (SEQ ID NO:9), Val-Val-Tyr-Pro-Trp-Thr-Gln(SEQ ID NO:10), Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:11),Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:12), Tyr-Pro-Trp-Thr-Gln (SEQ IDNO:13), and Tyr-Pro-Trp-Thr (SEQ ID NO:27).
 4. The method of claim 3,comprising contacting hematopoietic calls with a stem cell proliferationstimulating amount ofPhe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1). 5.The method of clam 3, comprising contacting hematopoietic cells with astem cell proliferation stimulating amount ofCys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) wherein the two Cys residues form a disulfide bond.
 6. A method asin claim 1 wherein said opiate compound is selected from the groupconsisting of morphine, etorphine, codeine, heroin, hydromorphone,oxymorphone, levorphanol, levallorphain, hydrocodone, oxycodone,nalorphine, naloxone, buprenorphine, butanorphanol, nalbuphine,meperidine, alphaprodine, diphenoxylate, fentanyl, (D-Ala²,N-Me-Phe⁴,glycin⁵)-Enkephalin, (D-Arg²,Lys⁴)-Dermorphin (1-4) amide andnociceptin.
 7. The method of claim 1, comprising contactinghematopoietic stem cells with a stem cell proliferation stimulatingamount of Phe-Pro-His-Pho-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ IDNO:1).
 8. The method of claim 1, comprising contacting hematopoieticstem cells with a stem cell proliferation stimulating amount ofCys-Phe-Pro-His-Pho-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) wherein the two Cys residues form a disulfide bond.
 9. A method ofstimulating stem cell proliferation comprising contacting hematopoieticcells with a stem cell proliferation stimulating amount of a compoundcapable of binding opiate receptors, wherein said stem cells are cellswhich can generate multiple lineages or other stem cells.
 10. A methodas in claim 9 wherein said compound has selectivity for the mu subclassof opiate receptor.
 11. A method of stimulating stem cell proliferationcomprising contacting stem cells with a stem cell proliferationstimulating amount of INPROL or an opiate compound or a stem cellproliferation stimulating amount of a combination at INPROL and anopiate compound, wherein sad INPROL is selected from the groupconsisting of a polypeptide having the sequence of amino acids 1-97 ofthe human alpha hemoglobin chain, a polypeptide having the sequence ofamino acids 1-94 of the human alpha hemoglobin chain,Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1),Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) (where the two Cys residues form a disuffide bond),Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(SEQ ID NO:3), Phe-Leu-Gly-Phe-Pro-Thr (SEQ ID NO:33),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:4),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:5),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:6),Leu-Val-Val-Tyr-Pro-Trp-Thr (SEQ ID NO:7), Leu-Val-Val-Tyr-Pro-Trp (SEQID NO:8), Leu-Val-Val-Tyr-Pro (SEQ ID NO:9), Val-Val-Tyr-Pro-Trp-Thr-Gln(SEQ ID NO:10), Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:11),Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:12), Tyr-Pro-Trp-Thr-Gln (SEQ IDNO:13), and Tyr-Pro-Trp-Thr (SEQ ID NO:27): wherein said stem cells arecells which can generate multiple lineages or other stem calls.
 12. Amethod as in claim 11 wherein said INPROL is selected from the groupconsisting of a polypeptide having the sequence of amino acids 1-97 ofthe human alpha hemoglobin chain, and a polypeptide having the sequenceof amino acids 1-94 of the human alpha hemoglobin chain.
 13. A method asin claim 11 wherein said INPROL is selected from the group consisting ofpeptides having the sequence:Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1),Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) (where the two Cys residues form a disuffide bond),Asp-Aa-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(SEQ NO:3), Phe-Leu-Gly-Phe-Pro-Thr (SEQ ID NO:33),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:4),Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:5), Lu-Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:6), Leu-Val-Val-Tyr-Pro-Trp-Thr(SEQ ID NO:7), Leu-Val-Val-Tyr-Pro-Trp (SEQ ID NO:8), Lu-Val-Val-Tyr-Pro (SEQ ID NO:9), Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ IDNO:10), Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:11),Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:12), Tyr-Pro-Trp-Thr-Gln (SEQ IDNO:13), and Tyr-Pro-Trp-Thr (SEQ ID NO:27).
 14. The method of claim 13,comprising contacting stem cells with a stem cell proliferationstimulating amount ofPhe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ ID NO:1).15. The method of claim 13, comprising contacting stem cells with a stemcell proliferation stimulating amount ofCys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ala-Gln-Val-Cys (SEQ ID NO:2)wherein the two Cys residues form a disuffide bond.